Methods and products for producing engineered mammalian cell lines with amplified transgenes

ABSTRACT

Methods of inserting genes into defined locations in the chromosomal DNA of cultured mammalian cell lines which are subject to gene amplification are disclosed. In particular, sequences of interest (e.g., genes encoding biotherapeutic proteins) are inserted proximal to selectable genes in amplifiable loci, and the transformed cells are subjected to selection to induce co-amplification of the selectable gene and the sequence of interest. The invention also relates to meganucleases, vectors and engineered cell lines necessary for performing the methods, to cell lines resulting from the application of the methods, and use of the cell lines to produce protein products of interest.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 16/258,207, filed Jan. 25, 2019, which is a continuation of U.S. patent application Ser. No. 15/783,243, filed Oct. 13, 2017, which is a continuation of U.S. patent application Ser. No. 14/806,175, filed Jul. 22, 2015, which is a continuation of U.S. patent application Ser. No. 14/091,572, filed Nov. 27, 2013, which is a continuation of International Application No. PCT/US2012/040599, filed Jun. 1, 2012, which claims priority to U.S. Provisional application No. 61/492,174 filed Jun. 1, 2011, the disclosures of all of which are hereby incorporated by reference in their entireties for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 13, 2022, is named P109070008US05-SEQ-NTJ.txt, and is 187,791 bytes in size.

FIELD OF THE INVENTION

The invention relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the invention relates to methods of inserting genes into defined locations in the chromosomal DNA of cultured mammalian cell lines which are subject to gene amplification. The invention also relates to meganucleases, vectors and engineered cell lines necessary for performing the methods, cell lines resulting from the application of the methods, and use of the cell lines to produce protein products of interest.

BACKGROUND OF THE INVENTION

Therapeutic proteins are the primary growth driver in the global pharmaceutical market (Kresse, Eur J Pharm Biopharm 72, 479 (2009)). In 2001, biopharmaceuticals accounted for $24.3 billion in sales. By 2007, this number had more than doubled to $54.5 billion. The market is currently estimated to reach $78 billion by 2012 (Pickering, Spectrum Pharmaceutical Industry Dynamics Report, Decision Resources, Inc., 5 (2008)). This includes sales of “blockbuster” drugs such as erythropoietin, tissue plasminogen activator, and interferon, as well as numerous “niche” drugs such as enzyme replacement therapies for lysosomal storage disorders. The unparalleled growth in market size, however, is driven primarily by skyrocketing demand for fully human and humanized monoclonal antibodies (Reichert, Curr Pharm Biotechnol 9, 423 (2008)). Because they have the ability to confer a virtually unlimited spectrum of biological activities, monoclonal antibodies are quickly becoming the most powerful class of therapeutics available to physicians. Not surprisingly, more than 25% of the molecules currently undergoing clinical trials in the United States and Europe are monoclonal antibodies (Reichert, Curr Pharm Biotechnol 9, 423 (2008)).

Unlike more traditional pharmaceuticals, therapeutic proteins are produced in living cells. This greatly complicates the manufacturing process and introduces significant heterogeneity into product formulations (Field, Recombinant Human IgG Production from Myeloma and Chinese Hamster Ovary Cells, in Cell Culture and Upstream Processing, Butler, ed., (Taylor and Francis Group, New York, 2007)). In addition, protein drugs are typically required at unusually high doses, which necessitates highly scalable manufacturing processes and makes manufacturing input costs a major price determinant. For these reasons, treatment with a typical therapeutic antibody (e.g., the anti-HER2-neu monoclonal Herceptin®) costs $60,000-$80,000 for a full course of treatment (Fleck, Hastings Center Report 36, 12 (2006)). Further complicating the economics of biopharmaceutical production is the fact that many of the early blockbuster biopharmaceuticals are off-patent (or will be off-patent soon) and the US and EU governments are expected to greatly streamline the regulatory approval process for “biogeneric” and “biosimilar” therapeutics (Kresse, Eur J Pharm Biopharm 72, 479 (2009)). These factors should lead to a significant increase in competition for sales of many prominent biopharmaceuticals (Pickering, Spectrum Pharmaceutical Industry Dynamics Report, Decision Resources, Inc., 5 (2008)). Therefore, there is enormous interest in technologies which reduce manufacturing costs of protein therapeutics (Seth et al., Curr Opin Biotechnol 18, 557 (2007)).

Many of the protein pharmaceuticals on the market are glycoproteins that cannot readily be produced in easy-to-manipulate biological systems such as bacteria or yeast. For this reason, recombinant therapeutic proteins are produced almost exclusively in mammalian cell lines, primarily Chinese hamster ovary (e.g., CHO-K1), mouse myeloma (e.g., NSO), baby hamster kidney (BHK), murine C127, human embryonic kidney (e.g., HEK-293), or human retina-derived (e.g., PER-C6) cells (Andersen and Krummen, Curr Opin Biotechnol 13, 117 (2002)). Of these, CHO cells are, by far, the most common platform for bioproduction because they offer the best combination of high protein expression levels, short doubling time, tolerance to a wide range of media conditions, established transfection and amplification protocols, an inability to propagate most human pathogens, a paucity of blocking intellectual property, and the longest track record of FDA approval (Field, Recombinant Human IgG Production from Myeloma and Chinese Hamster Ovary Cells, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)).

Large-market biopharmaceuticals are typically produced in enormous stirred-tank bioreactors containing hundreds of liters of CHO cells stably expressing the protein product of interest (Chu and Robinson, Curr Opin Biotechnol 12, 180 (2001), Coco-Martin and Harmsen, Bioprocess International 6, 28 (2008)). Under optimized industrial conditions, such manufacturing processes can yield in excess of 5 g of protein per liter of cells per day (Coco-Martin and Harmsen, Bioprocess International 6, 28 (2008)). Because of the large number of cells involved (˜50 billion cells per liter), the level of protein expression per cell has a very dramatic effect on yield. For this reason, all of the cells involved in the production of a particular biopharmaceutical must be derived from a single “high-producer” clone, the production of which constitutes one of the most time- and resource-intensive steps in the manufacturing process (Clarke and Compton, Bioprocess International 6, 24 (2008)).

The first step in the large-scale manufacture of a biopharmaceutical is the transfection of mammalian cells with plasmid DNA encoding the protein product of interest under the control of a strong constitutive promoter. Stable transfectants are selected by using a selectable marker gene also carried on the plasmid. Most frequently, this marker is a dihydrofolate reductase (DHFR) gene which, when transfected into a DHFR deficient cell line such as DG44, allows for the selection of stable transfectants using media deficient in hypoxanthine. The primary reason for using DHFR as a selectable marker is that it enables a process called “gene amplification”. By growing stable transfectants in gradually increasing concentrations of methotrexate (MTX), a DHFR inhibitor, it is possible to amplify the number of copies of the DHFR gene present in the genome. Because the gene encoding the protein product of interest is physically coupled to the DHFR gene, this results in amplification of both genes with a concomitant increase in the expression level of the therapeutic protein (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed., (Taylor and Francis Group, New York, 2007)). Related systems for the creation of stable bioproduction lines use the glutamine synthetase (GS) or hypoxanthine phosphoribosyltransferase (HPRT) genes as selectable markers and require the use of GS- or HPRT-deficient cell lines as hosts for transfection (Clarke and Compton, Bioprocess International 6, 24 (2008)). In the case of the GS system, gene amplification is accomplished by growing cells in the presence of methionine sulphoximine (MSX) (Clarke and Compton, Bioprocess International 6, 24 (2008)). In the case of the HPRT system, gene amplification is accomplished by growing cells in HAT medium, which contains aminopterin, hypoxanthine, and thymidine (Kellems, ed. Gene amplification in mammalian cells: a comprehensive guide, Marcel Dekker, New York, 1993).

In all of these systems, the initial plasmid DNA comprising a biotherapeutic gene expression cassette and a selectable marker integrates into a random location in the genome, resulting in extreme variability in therapeutic protein expression from one stable transfectant to another (Collingwood and Urnov , Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). For this reason, it is necessary to screen hundreds to thousands of initial transfectants to identify cells which express acceptably high levels of gene product both before and after gene amplification (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). A second and more problematic consequence of random gene integration is the phenomenon of transgene silencing, in which recombinant protein expression slows or ceases entirely over time (Collingwood and Urnov , Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Because these effects often do not manifest themselves for weeks to months following the initial transfection and screening process, it is generally necessary to carry and expand dozens of independent clonal lines to identify one that expresses the protein of interest consistently over time (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)).

This large number of screening and expansion steps results in a very lengthy and expensive process to simply generate the cell line that will, ultimately, produce the therapeutic of interest. Indeed, using conventional methods, a minimum of 10 months (with an average of 18 months) and an upfront investment of tens of millions of dollars in labor and material is required to produce an initial pool of protein-expressing cells suitable for industrial manufacturing (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). If one takes into account lost time on market for a blockbuster protein therapeutic, inefficiencies in cell line production can cost biopharmaceutical manufacturers hundreds of millions of dollars (Seth et al., Curr Opin Biotechnol 18, 557 (2007)).

Much of the time and expense of bioproduction cell line creation can be attributed to random genomic integration of the bioproduct gene resulting in clone-to-clone variability in genotype and, hence, variability in gene expression. One way to overcome this is to target gene integration to a defined location that is known to support a high level of gene expression. To this end, a number of systems have been described which use the Cre, Flp, or ΦC31 recombinases to target the insertion of a bioproduct gene (reviewed in Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Recent embodiments of these systems, most notably the Flp-In® system marketed by Invitrogen Corp. (Carlsbad, Calif.), couple bioproduct gene integration with the reconstitution of a split selectable marker so that cells with correctly targeted genes can be selected. As expected, these systems result in greatly reduced heterogeneity in gene expression and, in some cases, individual stable transfectants can be pooled, obviating the time and expense associated with expanding a single clone.

The principal drawback to recombinase-based gene targeting systems is that the recombinase recognition sites (loxP, FRT, or attB/attP sites) do not naturally occur in mammalian genomes. Therefore, cells must be pre-engineered to incorporate a recognition site for the recombinase before that site can be subsequently targeted for gene insertion. Because the recombinase site itself integrates randomly into the genome, it is still necessary to undertake extensive screening and evaluation to identify clones which carry the site at a location that is suitable for high level, long-term gene expression (Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). In addition, the biomanufacturing industry is notoriously hesitant to adopt “new” cell lines, such as those that have been engineered to carry a recombinase site, that do not have a track record of FDA approval. For these reasons, recombinase-based cell engineering systems may not readily be adopted by the industry and an approach that allows biomanufacturers to utilize their existing cell lines is preferable.

SUMMARY OF THE INVENTION

The present invention depends, in part, upon the development of mammalian cell lines in which sequences of interest (e.g., exogenous, actively transcribed transgenes) are inserted proximal to an endogenous selectable gene in an amplifiable locus, and the discovery that (a) the insertion of such exogenous sequences of interest does not inhibit amplification of the endogenous selectable gene, (b) the exogenous sequence of interest can be co-amplified with the endogenous selectable gene, and (c) the resultant cell lines, with an amplified region comprising multiple copies of the endogenous selectable gene and the exogenous sequence of interest, are stable for extended periods even in the absence of the selection regime which was employed to induce amplification. Thus, in one aspect, the invention provides a method for producing cell lines which can be used for biomanufacturing of a protein product of interest by specifically targeting the insertion of an exogenous sequence of interest capable of actively expressing the protein product of interest proximal to an endogenous selectable gene. In another aspect, the invention provides engineered cell lines that can be used to produce protein products of interest (e.g., therapeutic proteins such as monoclonal antibodies) at high levels.

It is understood that any of the embodiments described below can be combined in any desired way, and any embodiment or combination of embodiments can be applied to each of the aspects described below, unless the context indicates otherwise.

In one aspect, the invention provides a recombinant mammalian cell comprising an engineered target site stably integrated within selectable gene within an amplifiable locus, wherein the engineered target site disrupts the function of the selectable gene and wherein the engineered target site comprises a recognition sequence for a site specific endonuclease.

In some embodiments, the selectable gene is glutamine synthetase (GS) and the locus is methionine sulphoximine (MSX) amplifiable. In some embodiments, the selectable gene is dihydrofolate reductase (DHFR) and the locus is Methotrexate (MTX) amplifiable.

In some embodiments, the selectable gene is selected from the group consisting of Dihydrofolate Reductase, Glutamine Synthetase, Hypoxanthine Phosphoribosyltransferase, Threonyl tRNA Synthetase, Na,K-ATPase, Asparagine Synthetase, Ornithine Decarboxylase, Inosine-5′-monophosphate dehydrogenase, Adenosine Deaminase, Thymidylate Synthetase, Aspartate Transcarbamylase, Metallothionein, Adenylate Deaminase (1,2), UMP-Synthetase and Ribonucleotide Reductase.

In some embodiments, the selectable gene is amplifiable by selection with a selection agent selected from the group consisting of Methotrexate (MTX), Methionine sulphoximine (MSX), Aminopterin, hypoxanthine, thymidine, Borrelidin, Ouabain, Albizziin, Beta-aspartyl hydroxamate, alpha-difluoromethylornithine (DFMO), Mycophenolic Acid, Adenosine, Alanosine, 2′ deoxycoformycin, Fluorouracil, N-Phosphonacetyl-L-Aspartate (PALA), Cadmium, Adenine, Azaserine, Coformycin, 6-azauridine, pyrazofuran, hydroxyurea, motexafin gadolinium, fludarabine, cladribine, gemcitabine, tezacitabine and triapine.

In some embodiments, the engineered target site is inserted into an exon of the selectable gene. In some embodiments, the site specific endonuclease is a meganuclease, a zinc finger nuclease or TAL effector nuclease. In some embodiment, the recombinant cell further comprises the site specific endonuclease.

In one aspect, the invention provides a recombinant mammalian cell comprising an engineered target site stably integrated proximal to a selectable gene within an amplifiable locus, wherein the engineered target site comprises a recognition sequence for a site specific endonuclease.

In some embodiments, the engineered target site is downstream from the 3′ regulatory region of the selectable gene. In some embodiments, the engineered target site is 0 to 100,000 base pairs downstream from the 3′ regulatory region of the selectable gene. In other embodiments, the engineered target site is upstream from the 5′ regulatory region of the selectable gene. In some embodiments, the engineered target site is 0 to 100,000 base pairs upstream from the 5′ regulatory region of the selectable gene.

In another aspect, the invention provides a method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site proximal to a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5′ flanking region 5′ to the recognition sequence; and

(iii) a 3′ flanking region 3′ to the recognition sequence; and (b) introducing a double-stranded break between the 5′ and 3′ flanking regions of the endogenous target site; (c) contacting the cell with a donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the endogenous target site; (ii) an exogenous sequence; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the endogenous target site; whereby the donor 5′ flanking region, the exogenous sequence and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the endogenous target site by homologous recombination to provide a modified cell.

In some embodiments, the method futhter comprises growing the modified cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene. In some embodiments, the exogenous sequence comprises a gene of interest.

In some embodiments endogenous target site is downstream from the 3′ regulatory region of the selectable gene. In some embodiments, the endogenous target site is 0 to 100,000 base pairs downstream from the 3′ regulatory region of the selectable gene. In other embodiments, the endogenous target site is upstream from the 5′ regulatory region of the selectable gene. In some embodiments, the endogenous target site is 0 to 100,000 base pairs upstream from the 5′ regulatory region of the selectable gene.

In one aspect, the invention provides a method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site proximal to a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5′ flanking region 5′ to the recognition sequence; and (iii) a 3′ flanking region 3′ to the recognition sequence; and (b) introducing a double-stranded break between the 5′ and 3′ flanking regions of the endogenous target site; (c) contacting the cell with an engineered target site donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the endogenous target site; (ii) an exogenous sequence comprising an engineered target site; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the endogenous target site; whereby the donor 5′ flanking region, the exogenous sequence and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the endogenous target site by homologous recombination to provide a mammalian cell comprising the engineered target site; (d) introducing a double-stranded break between the 5′ and 3′ flanking regions of the engineered target site; (e) contacting the cell comprising the engineered target site with a sequence of interest donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the engineered target site; (ii) an exogenous sequence comprising a sequence of interest; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the engineered target site; whereby the donor 5′ flanking region, the exogenous sequence comprising the sequence of interest and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the engineered target site by homologous recombination to provide an engineered mammalian cell comprising the sequence of interest.

In some embodiments, the methof further comprises growing the engineered mammalian cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene. In some embodiments, the sequence of interest comprises a gene.

In another aspect, the invention provides a method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site within a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5′ flanking region 5′ to the recognition sequence; and

(iii) a 3′ flanking region 3′ to the recognition sequence; and (b) introducing a double-stranded break between the 5′ and 3′ flanking regions of the endogenous target site; (c) contacting the cell with an engineered target site donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the endogenous target site; (ii) an exogenous sequence comprising an engineered target site; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the endogenous target site; whereby the donor 5′ flanking region, the exogenous sequence and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the endogenous target site by homologous recombination to provide a mammalian cell comprising the engineered target site; (d) introducing a double-stranded break between the 5′ and 3′ flanking regions of the engineered target site; (e) contacting the cell comprising the engineered target site with a sequence of interest donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the engineered target site; (ii) an exogenous sequence comprising a sequence of interest; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the engineered target site; whereby the donor 5′ flanking region, the exogenous sequence comprising the sequence of interest and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the engineered target site by homologous recombination to provide a engineered mammalian cell comprising the sequence of interest.

In some emboduments, the method further comprises growing the engineered mammalian cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene.

In some embodiments, the sequence of interest comprises a gene.

In some embodiments, the endogenous target site is within an intron of the selectable gene. In other embodiments, the endogenous target site is within an exon of the selectable gene.

In one aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15.

In another aspect, the invention provides a recombinant meganuclease comprising the amino acid sequence of SEQ ID NO: 15.

In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 14.

In another aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9. In one embodiment, the recombinant meganuclease has the sequence of the meganuclease of SEQ ID NO: 9.

In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 7.

In another aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10. In one embodiment, the recombinant meganuclease comprises the polypeptide of SEQ ID NO: 10.

In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 8.

In another aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13. In one embodiment, the recombinant meganuclease comprises the polypeptide of SEQ ID NO: 13.

In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 12.

In another aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29. In one embodiment, the recombinant meganuclease comprises the polypeptide of SEQ ID NO: 29.

In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 30.

In another aspect, the invention provides recombinant mammalian cell lines which continue to express a protein product of interest from an exogenous sequence of interest present in an amplified region of the genome (i.e., present in 2-1,000 copies, co-amplified with a selectable gene in an amplifiable locus) for a period of at least 8, 9, 10, 11, 12, 13, or 14 weeks after removal of the amplification selection agent, and with a reduction of expression levels and/or copy number of less than 20, 25, 30, 35 or 40%.

In another aspect, the invention provides methods of producing recombinant cells with amplified regions including a sequence of interest and a selectable gene by subjecting the above-described recombinant cells to selection with a selection agent which causes co-amplification of the sequence of interest and the selectable gene.

In another aspect, the invention provides methods of producing a protein product of interest by culturing the above-described recombinant cells, or the above-described recombinant cells with amplified regions, and obtaining the protein product of interest from the culture medium or a cell lysate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A general strategy for targeting a sequence of interest to an amplifiable locus.

FIGS. 2A and 2B. (A) Schematic of the CHO DHFR locus showing a preferred region for targeting a sequence of interest 5,000-60,000 base pairs downstream of the DHFR gene. (B) Schematic of the CHO GS locus showing a preferred region for targeting a sequence of interest 5,000-55,000 base pairs downstream of the GS gene.

FIG. 3. Strategy for inserting a sequence of interest into an amplifiable locus in a two-step process involving a pre-integrated engineered target sequence.

FIG. 4. Strategy for inserting an engineered target sequence into an amplifiable locus with concomitant removal of a portion of the selectable gene, followed by insertion of a sequence of interest and reconstitution of the selectable gene.

FIG. 5. Strategy for inserting an engineered target sequence into an amplifiable locus with concomitant disruption of the coding sequence of a selectable gene, followed by insertion of a sequence of interest and reconstitution of the selectable gene.

FIG. 6. Strategy for inserting an engineered target sequence into an amplifiable locus with concomitant disruption of the mRNA processing, followed by insertion of a sequence of interest and reconstitution of the selectable gene.

FIGS. 7A through 7D. (A) A direct-repeat recombination assay for site-specific endonuclease activity. (B) Results of the assay in (A) applied to the CHO-23/24 and CHO-51/52 meganucleases.(C) Alignment of sequences obtained from CHO cells transfected with mRNA encoding the CHO-23/24 meganuclease (SEQ ID NOS 37-39, 38, 40, 38, and 38, respectively, in order of appearance). (D) Alignment of sequences obtained from CHO cells transfected with mRNA encoding the CHO-51/52 meganuclease (SEQ ID NOS 41-51, respectively, in order of appearance).

FIGS. 8A and 8B. (A) Strategy for inserting an exogenous DNA sequence into the CHO DHFR locus using the CHO-51/52 meganuclease.(B) PCR products demonstrating insertion of an engineered target sequence.

FIGS. 9A through 9C. (A) Strategy for inserting an engineered target sequence into the CHO DHFR locus using the CHO-23/24 meganuclease, followed by Flp recombinase-mediated insertion of a sequence of interest. (B) PCR products from hygromycin-resistant clones produced in (A). (C) GFP expression by the 24 clones produced in (B).

FIGS. 10A through 10C. Results of experiments with a GFP-expressing CHO line produced by integrating a GFP gene expression cassette into the DHFR locus using a target sequence strategy as shown in FIG. 9.

FIGS. 11A through 11C. (A) A direct-repeat recombination assay, as in FIG. 5A. (B) The assay in (A) applied to the CHO-13/14 and CGS-5/6 meganucleases. (C) Alignment of sequences obtained from CHO cells transfected with mRNA encoding the CGS-5/6 meganuclease (SEQ ID NOS 52-56, 56, 56-63, 63, 63, and 63-64, respectively, in order of appearance).

DETAILED DESCRIPTION OF THE INVENTION 1.1 Introduction

The present invention depends, in part, upon the development of mammalian cell lines in which exogenous actively transcribed transgenes have been inserted proximal to an endogenous amplifiable locus, and the discovery that (a) the insertion of such exogenous actively transcribed transgenes does not prevent or substantially inhibit amplification of the endogenous amplifiable locus, (b) the exogenous actively transcribed transgene can be co-amplified with the endogenous amplifiable locus, and (c) the resultant cell line, with an amplified region comprising multiple copies of the endogenous amplifiable locus and the exogenous actively transcribed transgene is stable for extended periods even in the absence of the selection regime which was employed to induce amplification. Thus, in one aspect, the invention provides a method for producing cell lines which can be used for biomanufacturing of a protein product of interest by specifically targeting the insertion of an exogenous gene capable of actively expressing the protein product of interest proximal to an endogenous amplifiable locus. In another aspect, the invention provides engineered cell lines that can be used to produce protein products of interest (e.g., therapeutic proteins such as monoclonal antibodies) at high levels.

1.2 References and Definitions

The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The entire disclosures of the issued U.S. patents, pending applications, published foreign applications, and scientific and technical references cited herein, including protein and nucleic acid database sequences, are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

As used herein, the term “meganuclease” refers to naturally-occurring homing endonucleases (also referred to as Group I intron encoded endonucleases) or non-naturally-occurring (e.g., rationally designed or engineered) endonucleases based upon the amino acid sequence of a naturally-occurring homing endonuclease. Examples of naturally-occurring meganucleases include I-SceI, I-CreI, I-CeuI, I-DmoI, I-MsoI, I-AniI, etc. Rationally designed meganucleases are disclosed in, for example, WO 2007/047859 and WO 2009/059195, and can be engineered to have modified DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties relative to a naturally occurring meganuclease. A meganuclease may bind to double-stranded DNA as a homodimer (e.g., wild-type I-CreI), or it may bind to DNA as a heterodimer (e.g., engineered meganucleases disclosed in WO 2007/047859). An engineered meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains derived from a natural meganuclease are joined into a single polypeptide using a peptide linker (e.g., single-chain meganucleases disclosed in WO 2009/059195).

As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of meganuclease subunits joined by a linker. A single-chain meganuclease has the organization: N-terminal subunit—Linker—C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will recognize non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. Methods of producing single-chain meganucleases are disclosed in WO 2009/059195.

As used herein, the term “site specific endonuclease” means a meganuclease, zinc-finger nuclease or TAL effector nuclease.

As used herein, with respect to a protein, the term “recombinant” means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the protein, and cells or organisms which express the protein. With respect to a nucleic acid, the term “recombinant” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant. As used herein, the term “engineered” is synonymous with the term “recombinant.”

As used herein, with respect to a meganuclease, the term “wild-type” refers to any naturally-occurring form of a meganuclease. The term “wild-type” is not intended to mean the most common allelic variant of the enzyme in nature but, rather, any allelic variant found in nature. Wild-type homing endonucleases are distinguished from recombinant or non-naturally-occurring meganucleases.

As used herein, the term “recognition sequence” refers to a DNA sequence that is bound and cleaved by a meganuclease. A recognition sequence comprises a pair of inverted, 9 base pair “half sites” which are separated by four base pairs. In the case of a homo- or heterodimeric meganucleases, each of the two monomers makes base-specific contacts with one half-site. In the case of a single-chain heterodimer meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. In the case if I-CreI, for example, the recognition sequence is 22 base pairs and comprises a pair of inverted, 9 base pair “half sites” which are separated by four base pairs.

As used herein, the term “target site” refers to a region of the chromosomal DNA of a cell comprising a target sequence into which a sequence of interest can be inserted. As used herein, the term “engineered target site” refers to an exogenous sequence of DNA integrated into the chromosomal DNA of a cell comprising an engineered target sequence into which a sequence of interest can be inserted.

As used herein, the term “target sequence” means a DNA sequence within a target site which includes one or more recognition sequences for a nuclease, integrase, transposase, and/or recombinase. For example, a target sequence can include a recognition sequence for a meganuclease. As used herein, an “engineered target sequence” means an exogenous target sequence which is introduced into a chromosome to serve as the insertion point for another sequence.

As used herein, the term “flanking region” or “flanking sequence” refers to a sequence of >3 or, preferably, >50 or, more preferably, >200 or, most preferably, >400 base pairs of DNA which is immediately 5′ or 3′ to a reference sequence (e.g., a target sequence or sequence of interest).

As used herein, the terms “amplifiable locus” refers to a region of the chromosomal DNA of a cell which can be amplified by selection with one or more compounds (e.g., drugs) in the growth media. An amplifiable locus will typically comprise a gene encoding a protein which, under the appropriate conditions, is necessary for cell survival. By inhibiting the function of such an essential protein, for example with a small molecule drug, the amplifiable locus is duplicated many times over as a means of increasing the copy number of the essential gene. A gene of interest, if integrated into an amplifiable locus, will also become duplicated with the essential gene. Examples of amplifiable loci include the chromosomal regions comprising the DHFR, GS, and HPRT genes.

As used herein, the term “amplified locus” or “amplified gene” or “amplified sequence” refers to a locus, gene or sequence which is present in 2-1,000 copies as a result of gene amplification in response to selection of a selectable gene. An amplified gene or sequence can be a gene or sequence which is co-amplified due to selection of a selectable gene in the same amplifiable locus. In preferred embodiments, a sequence of interest is amplified to at least 3, 4, 5, 6, 7, 8, 9 or 10 copies.

As used herein, the term “selectable gene” refers to an endogenous gene that is essential for cell survival under some specific culture conditions (e.g., presence or absence of a nutrient, toxin or drug). Selectable genes are endogenous to the cell and are distinguished from exogenous “selectable markers” such as antibiotic resistance genes. Selectable genes exist in their natural context in the chromosomal DNA of the cell. For example, DHFR is a selectable gene which is necessary for cell survival in the presence of MTX in the culture medium. The gene is essential for growth in the absence of hypoxanthine and thymidine. If the endogenous DHFR selectable gene is eliminated, cells are able to grow in the absence of hypoxanthine and thymidine if they are given an exogenous copy of the DHFR gene. This exogenous copy of the DHFR gene is a selectable marker but is not a selectable gene. An amplifiable locus comprises a selectable gene and a target site. A target site is found outside of a selectable gene such that a selectable gene does not comprise a target site. Examples of selectable genes are given in Table 1.

As used herein, when used in connection with the position of a target site, recognition sequence, or inserted sequence of interest relative to the position of a selectable gene, the term “proximal” means that the target site, recognition sequence, or inserted sequence of interest is within the same amplifiable locus as the selectable gene, either upstream (5′) or downstream (3′) of the selectable gene, and preferably between the selectable gene and the next gene in the region (whether upstream (5′) or downstream (3′)). Typically, a “proximal” target site, recognition sequence, or inserted sequence of interest will be within <100,000 base pairs of the selectable gene, as measured from the first or last nucleotide of the first or last regulatory element of the selectable gene.

As used herein, the term “homologous recombination” refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell. Thus, for some applications of engineered meganucleases, a meganuclease is used to cleave a recognition sequence within a target sequence in a genome and an exogenous nucleic acid with homology to or substantial sequence similarity with the target sequence is delivered into the cell and used as a template for repair by homologous recombination. The DNA sequence of the exogenous nucleic acid, which may differ significantly from the target sequence, is thereby inserted or incorporated into the chromosomal sequence. The process of homologous recombination occurs primarily in eukaryotic organisms. The term “homology” is used herein as equivalent to “sequence similarity” and is not intended to require identity by descent or phylogenetic relatedness.

As used herein, the term “stably integrated” means that an exogenous or heterologous DNA sequence has been covlently inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transposition, etc.) and has remained in the chromosome for a period of at least 8 weeks.&&

As used herein, the term “non-homologous end-joining” or “NHEJ” refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. Thus, for certain applications, an engineered meganuclease can be used to produce a double-stranded break at a meganuclease recognition sequence within an amplifiable locus and an exogenous nucleic acid molecule, such as a PCR product, can be captured at the site of the DNA break by NHEJ (see, e.g. Salomon et al. (1998), EMBO J. 17:6086-6095). In such cases, the exogenous nucleic acid may or may not have homology to the target sequence. The process of non-homologous end-joining occurs in both eukaryotes and prokaryotes such as bacteria.

As used herein, the term “sequence of interest” means any nucleic acid sequence, whether it codes for a protein, RNA, or regulatory element (e.g., an enhancer, silencer, or promoter sequence), that can be inserted into a genome or used to replace a genomic DNA sequence. Sequences of interest can have heterologous DNA sequences that allow for tagging a protein or RNA that is expressed from the sequence of interest. For instance, a protein can be tagged with tags including, but not limited to, an epitope (e.g., c-myc, FLAG) or other ligand (e.g., poly-His). Furthermore, a sequence of interest can encode a fusion protein, according to techniques known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley 1999). In preferred embodiments, a sequence of interest comprises a promoter operably linked to a gene encoding a protein of medicinal value such as an antibody, antibody fragment, cytokine, growth factor, hormone, or enzyme. For some applications, the sequence of interest is flanked by a DNA sequence that is recognized by the engineered meganuclease for cleavage. Thus, the flanking sequences are cleaved allowing for proper insertion of the sequence of interest into genomic recognition sequences cleaved by an engineered meganuclease. For some applications, the sequence of interest is flanked by DNA sequences with homology to or substantial sequence similarity with the target site such that homologous recombination inserts the sequence of interest within the genome at the locus of the target sequence.

As used herein, the term “donor DNA” refers to a DNA molecule comprising a sequence of interest flanked by DNA sequences homologous to a target site. Donor DNA can serve as a template for DNA repair by homologous recombination if it is delivered to a cell with a site-specific nuclease such as a meganuclease, zinc-finger nuclease, or TAL-effector nuclease. The result of such DNA repair is the insertion of the sequence of interest into the chromosomal DNA of the cell. Donor DNA can be linear, such as a PCR product, or circular, such as a plasmid. In cases where a donor DNA is a circular plasmid, it may be referred to as a “donor plasmid.”

As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or.”

2.1 Transgene Targeting to Amplifiable Loci

The present invention provides methods for generating transgenic mammalian cell lines expressing a desired protein product of interest, including “high-producer” cell lines, by targeting the insertion of a gene encoding the protein product of interest (e.g., a therapeutic protein gene expression cassette) to regions of the genome that are amplifiable. Such regions in mammalian cells include the DHFR, GS, and HPRT genes, as well as others shown in Table 1.

The precise mechanism of gene amplification is not known. Indeed, it is very likely that there is no single mechanism by which gene amplification occurs but that a variety of different random chromosomal aberrations, in combination with strong selection for amplification, results in increased gene copy number (reviewed in Omasa (2002), J. Biosci. Bioeng. 94:600-605). It is clear that chromosomal location plays a major role in amplification and the stable maintenance of amplified genes (Brinton and Heintz (1995), Chromosoma 104:143-51). It has been found that transgenes integrated into chromosomal locations adjacent to telomeres are more easily amplified and, once amplified, tend to be stable at high copy numbers after the selection agent is removed (Yoshikawa et al. (2000), Cytotechnology 33:37-46; Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715). This is significant because selection agents such as MTX and MSX are toxic and cannot be included in the growth media in a commercial biomanufacturing process. In contrast, transgenes integrated into regions in the CHO genome that are not adjacent to telomeres amplify inefficiently and rapidly lose copy number following the removal of selection agents from the media. For example, Yoshikawa et al. found that randomly-integrated transgenes linked to a DHFR selectable marker amplified to greater than 10-fold higher copy numbers when the integration site was adjacent to a telomere (Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715). These researchers also found that an amplified transgene integrated into a non-telomeric region will lose >50% of its copies in only 20 days following the removal of MTX from the growth media. None of the selectable genes identified in Table 1 is adjacent to a telomere in the mouse genome (www.ensembl.com) and the similarity in genome organization between mouse and CHO makes it likely that these genes are in non-telomeric regions in CHO as well (Xu et al. (2011), Nat. Biotechnol. 29:735-741). Thus, the prior art instructs that the loci identified in Table 1, including the DHFR and GS loci, are not preferred locations to target transgene insertion if the goal is efficient and stable gene amplification.

In addition, in the case of endogenous gene amplification, it is clear that chromosomal sequences outside of the selectable gene sequence play an important role in facilitating amplification and in defining the length of DNA sequence that is co-amplified with the gene under selection (Looney and Hamlin (1987), Mol. and Cell. Biol. 7:569-577). In particular, it has been shown that the sequence and location of the DNA replication origin in relation to the selectable gene plays a major role in amplification. For example, it has been shown that amplification of the endogenous CHO DHFR locus is dependent upon a pair of replication origins found in the region 5,000-60,000 base pairs downstream of the DHFR gene coding sequence (Anachkova and Hamlin (1989), Mol. and Cell. Biol. 9:532-540; Milbrandt et al. (1981), Proc. Natl. Acad. Sci. USA 78:6042-6047). Further, Brinton and Heintz have shown that these same replication origins fail to promote gene amplification when incorporated randomly into the genome with a transgenic DHFR sequence (Brinton and Heintz (1995), Chromosoma. 104:143-51). This clearly demonstrates the importance of maintaining both the sequence and proper chromosomal context of these replication origins to promote DHFR gene amplification. Thus the art instructs that the region downstream of DHFR is critical to gene amplification and should not be disrupted by, for example, inserting a transgenic gene expression cassette as described in the present invention.

Surprisingly, we have discovered that DNA sequences, including exogenous transcriptionally active sequences, which are inserted proximal to (e.g., within <100,000 base pairs) selectable genes in mammalian cell lines (e.g., CHO-K1) will co-amplify in the presence of appropriate compounds which select for amplification. Thus, the present invention provides methods for reliably and reproducibly producing isogenic cell lines in which transgenes encoding protein products of interest (e.g., biotherapeutic gene expression cassettes) can be amplified but in which it is not necessary to screen a large number of randomly generated cell lines to identify those which express high levels of the protein product of interest and are resistant to gene silencing.

In addition, we have surprisingly found that the mammalian cell lines of the invention, in which a sequence of interest is co-amplified with a selectable gene in an amplifiable locus, are stable with respect to expression of the sequence of interest and/or copy number of the sequence of interest even in the absence of continued selection. That is, whereas the art teaches that amplified sequences will be reduced in copy number over time if selection is not maintained (see, e.g., Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715), we have found that cell lines produced according to the methods of the invention continue to produce the protein products of interest (encoded by the sequences of interest) at levels within 20%-25% of the initial levels, even 14 weeks after removal of the selection agent. This is significant, as noted above, because selection agents such as MTX and MSX are toxic, and it would be highly desirable to produce biotherapeutic proteins in cell lines which do not require continued exposure to such selection agents. Therefore, in some embodiments, the invention provides recombinant mammalian cell lines which continue to express a protein product of interest from an exogenous sequence of interest present in an amplified region of the genome (i.e., present in 2-1,000 copies, co-amplified with a selectable gene in an amplifiable locus) for a period of at least 8, 9, 10, 11, 12, 13, or 14 weeks after removal of the amplification selection agent, and with a reduction of expression levels and/or copy number of less than 20, 25, 30, 35 or 40%.

The present invention also provides the products necessary to practice the methods, and to target insertion of sequences of interest into amplifiable loci in mammalian cell lines. A common method for inserting or modifying a DNA sequence involves introducing a transgenic DNA sequence flanked by sequences homologous to the genomic target and selecting or screening for a successful homologous recombination event. Recombination with the transgenic DNA occurs rarely but can be stimulated by a double-stranded break in the genomic DNA at the target site (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73; Tzfira et al. (2005), Trends Biotechnol. 23: 567-9; McDaniel et al. (2005), Curr. Opin. Biotechnol. 16: 476-83). Numerous methods have been employed to create DNA double-stranded breaks, including irradiation and chemical treatments. Although these methods efficiently stimulate recombination, the double-stranded breaks are randomly dispersed in the genome, which can be highly mutagenic and toxic. At present, the inability to target gene modifications to unique sites within a chromosomal background is a major impediment to routine genome engineering.

One approach to achieving this goal is stimulating homologous recombination at a double-stranded break in a target locus using a nuclease with specificity for a sequence that is sufficiently large to be present at only a single site within the genome (see, e.g., Porteus et al. (2005), Nat. Biotechnol. 23: 967-73). The effectiveness of this strategy has been demonstrated in a variety of organisms using ZFNs (Porteus (2006), Mol Ther 13: 438-46; Wright et al. (2005), Plant J. 44: 693-705; Urnov et al. (2005), Nature 435: 646-51). Homing endonucleases are a group of naturally-occurring nucleases which recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as Group I self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double-stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases are commonly grouped into four families: the LAGLIDADG (SEQ ID NO: family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLIDADG (SEQ ID NO: 65) family are characterized by having either one or two copies of the conserved LAGLIDADG (SEQ ID NO: 65) motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757-3774). The LAGLIDADG (SEQ ID NO: 65) homing endonucleases with a single copy of the LAGLIDADG (SEQ ID NO: 65) motif form homodimers, whereas members with two copies of the LAGLIDADG (SEQ ID NO: 65) motif are found as monomers.

Natural homing endonucleases, primarily from the LAGLIDADG (SEQ ID NO: 65) family, have been used to effectively promote site-specific genome modification in plants, yeast, Drosophila, mammalian cells and mice, but this approach has been limited to the modification of either homologous genes that conserve the endonuclease recognition sequence (Monnat et al. (1999), Biochem. Biophys. Res. Commun. 255: 88-93) or to pre-engineered genomes into which a recognition sequence has been introduced (Rouet et al. (1994), Mol. Cell. Biol. 14: 8096-106; Chilton et al. (2003), Plant Physiol. 133: 956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5055-60; Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al. (2006), J. Gene Med. 8(5):616-622).

Systematic implementation of nuclease-stimulated gene modification requires the use of engineered enzymes with customized specificities to target DNA breaks to existing sites in a genome and, therefore, there has been great interest in adapting homing endonucleases to promote gene modifications at medically or biotechnologically relevant sites (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73; Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62).

I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 65) family of homing endonucleases which recognizes and cuts a 22 base pair recognition sequence in the chloroplast chromosome of the algae Chlamydomonas reinhardtii. Genetic selection techniques have been used to modify the wild-type I-CreI cleavage site preference (Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Chames et al. (2005), Nucleic Acids Res. 33: e178; Seligman et al. (2002), Nucleic Acids Res. 30: 3870-9, Arnould et al. (2006), J. Mol. Biol. 355: 443-58). More recently, a method of rationally-designing mono-LAGLIDADG (SEQ ID NO: 65) homing endonucleases was described which is capable of comprehensively redesigning I-CreI and other homing endonucleases to target widely-divergent DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes (WO 2007/047859).

Thus, in one embodiment, the invention provides engineered meganucleases derived from the amino acid sequence of I-CreI that recognize and cut DNA sites in amplifiable regions of mammalian genomes. These engineered meganucleases can be used in accordance with the invention to target the insertion of gene expression cassettes into defined locations in the chromosomal DNA of cell lines such as CHO cells. This invention will greatly streamline the production of desired cell lines by reducing the number of lines that must be screened to identify a “high-producer” clone suitable for commercial-scale production of a therapeutic glycoprotein.

The present invention involves targeting transgenic DNA “sequences of interest” to amplifiable loci. The amplifiable loci are regions of the chromosomal DNA that contain selectable genes that become amplified in the presence of selection agents (e.g., drugs). For example, the Chinese Hamster Ovary (CHO) cell DHFR locus can be amplified to ˜1,000 copies by growing the cells in the presence of methotrexate (MTX), a DHFR inhibitor. Table 1 lists additional examples of selectable genes that can be amplified using small molecule drugs (Kellems, ed. Gene amplification in mammalian cells: a comprehensive guide. Marcel Dekker, New York, 1993; Omasa (2002), J. Biosci. Bioeng. 94:6 600-605).

TABLE 1 Amplifiable Genes Selectable Gene Name Amplified With Dihydrofolate Reductase Methotrexate (MTX) Glutamine Synthetase Methionine sulphoximine (MSX) Hypoxanthine Aminopterin, hypoxanthine, and Phosphoribosyltransferase thymidine Threonyl tRNA Synthetase Borrelidin Na,K-ATPase Ouabain Asparagine Synthetase Albizziin or Beta-aspartyl hydroxamate Ornithine Decarboxylase alpha-difluoromethylornithine (DFMO) Inosine-5′-monophosphate Mycophenolic Acid dehydrogenase Adenosine Deaminase Adenosine, Alanosine, 2′deoxycoformycin Thymidylate Synthetase Fluorouracil Aspartate Transcarbamylase N-Phosphonacetyl-L-Aspartate (PALA) Metallothionein Cadmium Adenylate Deaminase (1,2) Adenine, Azaserine, Coformycin UMP-Synthetase 6-azauridine, pyrazofuran Ribonucleotide Reductase hydroxyurea, motexafin gadolinium, fludarabine, cladribine, gemcitabine, tezacitabine, triapine.

Several considerations must be taken into account when selecting a specific target site for the insertion of a sequence of interest within an amplifiable locus. First, the selected insertion site must be co-amplified with the gene under selection. In many cases, experimental data already exists in the art which delimits the amount of flanking chromosomal sequence that co-amplifies with a selectable gene of interest. This data, which precisely defines the extent of the amplifiable locus, exists for CHO DHFR (Ma et al. (1988), Mol Cell Biol. 8(6):2316-27), human DHFR (Morales et al. (2009), Mol Cancer Ther. 8(2):424-432), and CHO GS (Sanders et al. (1987), Dev Biol Stand. 66:55-63). Where such data does not already exist in the art, we predict that chromosomal DNA sequences <100,000 base pairs upstream or downstream of the selectable gene coding sequence are likely to co-amplify. Hence, these regions could be suitable sites for targeting the insertion of a sequence of interest.

Second, target sites should be selected which will not greatly impact the function of the selectable gene (e.g., the endogenous DHFR, GS, or HPRT gene). Because amplification requires a functional copy of the selectable gene, insertion sites within the promoter, exons, introns, polyadenylation signals, or other regulatory sequences that, if disrupted, would greatly impact transcription or translation of the selectable gene, should be avoided. For example, WO 2008/059317 discloses meganucleases which cleave DNA target sites within the HPRT gene. To the extent WO 2008/059317 discloses the insertion of genes into the HPRT locus, it teaches that the HPRT gene coding sequence should be disrupted in the process of transgene insertion to facilitate selection for proper targeting using 6-thioguanine. 6-thioguanine is a toxic nucleotide analog that kills cells having functional HPRT activity. Because cells produced in accordance with WO 2008/059317 will not have HPRT activity, they will not amplify an inserted transgene in response to treatment with an HPRT inhibitor and, so, cannot be used in the present invention. For the present invention, unless the precise limits of all regulatory sequences are already known for a particular selectable gene, insertion sites >1,000 base pairs, >2,000 base pairs, >3,000 base pairs, >4,000 base pairs, or, preferably, >5,000 base pairs, upstream or downstream of the gene coding sequence should be selected. However, if the location of the regulatory sequences are known, the sequence of interest can be inserted immediately adjacent to the either the most 5′ or 3′ regulatory sequence (e.g., immediately 3′ to the polyadenylation signal).

Lastly, target sites should be selected which do not disrupt other chromosomal genes which may be important for normal cell physiology. In general, gene insertion sites should be >1,000 base pairs, >2,000 base pairs, >3,000 base pairs, >4,000 base pairs, or, preferably, >5,000 base pairs, away from any gene coding sequence.

Various methods of the invention are described schematically in the figures as follows:

FIG. 1 depicts a general strategy for targeting a sequence of interest to an amplifiable locus. In the first step, a site-specific endonuclease introduces a double-stranded break in the chromosomal DNA of a cell at a site that is proximal to an endogenous selectable gene. The cleaved chromosomal DNA then undergoes homologous recombination with a donor DNA molecule comprising a sequence of interest flanked by DNA sequences homologous to sequences flanking the endonuclease recognition sequence in the target site. As a result, the sequence of interest is inserted into the chromosomal DNA of the cell adjacent to the endogenous selectable gene. The modified cell is then grown in the presence of one or more compounds that inhibit the function of the selectable gene to induce an increase in the copy number (i.e., amplification) of the selectable gene. The sequence of interest, which is genetically linked to the selectable gene, will co-amplify with the selectable gene. The result is a stable transgenic cell line comprising multiple copies of the sequence of interest.

FIG. 2(A) depicts a schematic of the CHO DHFR locus showing a preferred region for targeting a sequence of interest 5,000-60,000 base pairs downstream of the DHFR gene. FIG. 2(B) depicts a schematic of the CHO GS locus showing a preferred region for targeting a sequence of interest 5,000-55,000 base pairs downstream of the GS gene. Promoters are shown as arrows. Exons are shown as rectangles, with non-coding exons in white and protein coding exons in gray.

FIG. 3 depicts a strategy for inserting a sequence of interest into an amplifiable locus in a two-step process involving a pre-integrated target sequence. In the first step, the chromosomal DNA of a cell is cleaved by a site-specific endonuclease at a site that is proximal to a selectable gene. The cleaved chromosomal DNA then undergoes homologous recombination with a donor DNA molecule comprising an exogenous target sequence flanked by DNA sequences homologous to the sequences flanking the endogenous target site. This results in the insertion of the new engineered target sequence into the chromosomal DNA of the cell proximal to the selectable gene. A sequence of interest can subsequently be targeted proximal to the same selectable gene using a nuclease, integrase, transposase, or recombinase that specifically recognizes the pre-integrated engineered target sequence. The modified cell is then grown in the presence of one or more compounds that co-amplify the selectable gene and the sequence of interest.

FIG. 4 depicts a strategy for inserting an engineered target sequence into a selectable gene (e.g., DHFR) with concomitant removal of a portion of the selectable gene. A site-specific endonuclease is first used to cleave the chromosomal DNA of the cell proximal to or within the selectable gene sequence. As shown in the figure, the endogenous target site is between exons 2 and 3 of the CHO DHFR gene (although the target site could be within any intron or exon, and the selectable gene could be any gene subject to amplification). The chromosomal DNA then undergoes homologous recombination with a first donor DNA (“donor DNA #1”) such that the sequence of the first donor DNA is inserted into the chromosomal DNA of the cell. As shown in the figure, this results in the replacement of the promoter and first two exons of DHFR by the new engineered target sequence (although the first donor DNA could replace more or less of the chromosomal DNA, such as only a portion of one exon). If such a replacement is made to all DHFR alleles in a cell, the resultant cell line is DHFR (−/−). A sequence of interest can subsequently be targeted proximal to the selectable gene in the cell line using an endonuclease, integrase, transposase, or recombinase that recognizes the engineered target sequence. As shown in the figure, the second donor DNA (“donor DNA #2”) comprises a sequence of interest as well as a promoter and the first two exons of DHFR. Proper targeting of this second donor DNA molecule results in the insertion of the sequence of interest at the engineered target sequence while simultaneously reconstituting a functional DHFR gene. Thus, properly targeted cell lines will be DHFR+ and can be selected using media deficient in hypoxanthine/thymidine. In addition, the sequence of interest can be co-amplified with the DHFR gene using MTX selection. The strategy diagrammed here for DHFR can be applied to any selectable gene in an amplifiable locus.

FIG. 5 depicts a strategy for inserting an engineered target sequence into an amplifiable locus with concomitant disruption of the coding sequence of a selectable gene. A site-specific endonuclease is first used to cleave the chromosomal DNA of the cell within the selectable gene coding sequence. As shown in the figure, the endogenous target site is in the third exon of the CHO GS gene. The chromosomal DNA then undergoes homologous recombination with a first donor DNA (“donor DNA #1”) such that the sequence of the first donor DNA is inserted into the chromosomal DNA of the cell. This results in the insertion of a new engineered target sequence into the GS coding sequence. If such an insertion occurs in both alleles of the GS gene and results in a frameshift mutation or otherwise disrupts the function of the GS gene, the resultant cell line will be GS (−/−). A sequence of interest can subsequently be targeted proximal to the amplifiable locus in the cell line using an endonuclease, integrase, transposase, or recombinase that recognizes the engineered target sequence. As shown in the figure, a second donor DNA (“donor DNA #2”) comprises a sequence of interest operably linked to a promoter as well as the 3′ portion of the GS coding sequence comprising exons 3, 4, 5, and 6. (The figure shows exons 3, 4, 5, and 6 joined into a single nucleotide sequence (i.e., with introns removed), but a sequence including either the naturally-occurring introns or one or more artificial introns could also be employed). Proper targeting of the second donor DNA molecule results in the insertion of the sequence of interest at the engineered target sequence while simultaneously reconstituting a functional GS gene. Thus, properly targeted cell lines will be GS+ and can be selected using media deficient in L-glutamine. In addition, the sequence of interest can be co-amplified with the GS gene using MSX selection. The strategy diagrammed here for GS can be applied to any selectable gene in an amplifiable locus.

FIG. 6 depicts a strategy for inserting an engineered target sequence into an amplifiable locus with concomitant disruption of the mRNA processing of a selectable gene. A site-specific endonuclease is first used to cleave the chromosomal DNA of the cell within an intron in the selectable gene. As drawn, the endogenous target site is in the intron between the third and fourth coding exons of the CHO GS gene. The chromosomal DNA then undergoes homologous recombination with a donor DNA #1 such that the sequence of the donor DNA is inserted in the chromosomal DNA of the cell. This results in the insertion of a new engineered target sequence into the GS coding sequence with an additional sequence that causes the GS mRNA to be processed incorrectly. As drawn, this additional sequence comprises a strong splice acceptor. If such an insertion occurs in both alleles of the GS gene, the artificial splice acceptor will cause the GS mRNA to splice incorrectly, resulting in a loss of GS expression and a requirement for growth in media containing L-glutamine. A sequence of interest can subsequently be targeted to the amplifiable locus in the cell line using an endonuclease, integrase, transposase, or recombinase that recognizes the engineered target sequence. As diagrammed, donor DNA #2 comprises a sequence of interest operably linked to a promoter as well as the 3′ portion of the GS coding sequence comprising exons 4, 5, and 6 joined into a single nucleotide sequence. (The figure shows exons 4, 5, and 6 joined into a single nucleotide sequence (i.e., with introns removed), but a sequence including either the naturally-occurring introns or one or more artificial introns could also be employed). Proper targeting of this donor DNA #2 molecule results in the insertion of the sequence of interest at the engineered target sequence while simultaneously reconstituting a functional GS gene. Thus, properly targeted cell lines will be GS+and can be selected using media deficient in L-glutamine and the sequence of interest can be co-amplified with the GS gene using MSX selection. The strategy diagrammed here for GS can be applied to any selectable gene in an amplifiable locus.

FIG. 7(A) depicts a direct-repeat recombination assay for site-specific endonuclease activity. A reporter plasmid is produced comprising the 5′ two-thirds of the GFP gene (“GF”), followed by an endonuclease recognition sequence, followed by the 3′ two-thirds of the GFP gene (“FP”). Mammalian cells are transfected with this reporter plasmid as well as a gene encoding an endonuclease. Cleavage of the recognition sequence by the endonuclease stimulates homologous recombination between direct repeats of the GFP gene to restore GFP function. GFP+ cells can then be counted and/or sorted on a flow cytometer.

FIG. 7(B) depicts the results of the assay of FIG. 7(A) as applied to the CHO-23/24 and CHO-51/52 meganucleases. Light bars indicate the percentage of GFP+ cells when cells are transfected with the reporter plasmid alone (−endonuclease). Dark bars indicate the percentage of GFP+ cells when cells are co-transfected with a reporter plasmid and the corresponding meganuclease gene (+endonuclease). The assay was performed in triplicate and the standard deviation is shown.

FIG. 7(C) depicts alignment of sequences obtained from CHO cells transfected with mRNA encoding the CHO-23/24 meganuclease. The top sequence is from a wild-type (WT) CHO cell with the recognition sequence for CHO-23/24 underlined.

FIG. 7(D) depicts alignment of sequences obtained from CHO cells transfected with mRNA encoding the CHO-51/52 meganuclease. The top sequence is from a wild-type (WT) CHO cell with the recognition sequence for CHO-51/52 underlined.

FIG. 8(A) depicts a strategy for inserting an exogenous DNA sequence into the CHO DHFR locus using the CHO-51/52 meganuclease. CHO cells were co-transfected with mRNA encoding CHO-51/52 and a donor plasmid comprising an EcoRI site flanked by 543 base pairs of DNA sequence homologous to the region upstream of the CHO-51/52 recognition site and 461 base pairs of DNA sequence homologous to the region downstream of the CHO-51/52 recognition site. 48 hours post-transfection, genomic DNA was isolated and subjected to PCR using primers specific for the downstream region of the DHFR locus (dashed arrows).

FIG. 8(B) depicts PCR products that were cloned into pUC-19 and 48 individual plasmid clones and were digested with EcoRI and visualized on an agarose gel. 10 plasmids (numbered lanes) yielded a 647 base pair restriction fragment, consistent with cleavage of a first EcoRI site within the pUC-19 vector and a second EcoRI site in the cloned PCR fragment. These 10 plasmids were sequenced to confirm that they harbor a PCR fragment comprising a portion of the downstream DHFR locus with an EcoRI restriction site inserted into the CHO-51/52 recognition sequence. This restriction pattern was not observed when CHO cells were transfected with the donor plasmid alone.

FIG. 9(A) depicts a strategy for inserting an engineered target sequence into the CHO DHFR locus using the CHO-23/24 meganuclease. CHO cells were co-transfected with mRNA encoding CHO-23/24 and a donor plasmid comprising, in 5′ to 3′ orientation, an SV40 promoter, an ATG start codon, an FRT site, and a Zeocin-resistance (Zeo) gene. Zeocin-resistant cells were cloned by limiting dilution and screened by PCR to identify a clonal cell line in which the donor plasmid sequence integrated into the CHO-23/24 recognition site. After expansion, this cell line was co-transfected with a first plasmid encoding Flp recombinase operably linked to a promoter and second plasmid (donor plasmid #2) comprising a GFP gene under the control of a CMV promoter, an FRT site, and a hygromycin-resistance (Hyg) gene lacking a start codon. Flp-mediated recombination between FRT sites resulted in the integration of the donor plasmid #2 sequence into the engineered target sequence (i.e., the FRT site) such that a functional Hyg gene expression cassette was produced. FIG. 9(B) depicts PCR products from hygromycin-resistant clones produced as in (A) that were cloned by limiting dilution. Genomic DNA was extracted from 24 individual clones and PCR amplified using a first primer in the DHFR locus and a second primer in the Hyg gene (dashed lines). All 24 clones yielded a PCR product consistent with Hyg gene insertion into the engineered target sequence. FIG. 9(C) depicts GFP expression by the 24 clones produced in (B) using flow cytometry. All clones were found to express high levels of GFP with relatively little clone-to-clone variability.

FIG. 10. A GFP-expressing CHO line was produced by integrating a GFP gene expression cassette into the DHFR locus using an engineered target sequence strategy as shown in FIG. 9. This cell line was then grown in MTX as described in Example 2 to amplify the integrated GFP gene. (A) Flow cytometry plots showing GFP intensity on the Y-axis. In the pre-MTX cell line, GFP intensity averages approximately 2×10³ whereas in the cell line grown in 250 nM MTX, a distinct sub-population is visible (circled) in which GFP intensity approaches 10⁴. (B) MTX treated cell lines were sorted by FACS to identify individual cells expressing higher amounts of GFP. Five such high-expression cells were expanded and GFP intensity was determined by flow cytometry. All five clones were found to have significantly increased GFP expression relative to the pre-MTX cell line. (C) Genomic DNA was isolated from the five clonal cell lines produced in (B) and subjected to quantitative PCR using a primer pair specific for the GFP gene. It was found that the five high-expression clones had significantly more copies of the GFP gene than the pre-MTX cell line. These results demonstrate that the copy number and expression level a transgene integrated downstream of CHO DHFR can amplify in response to MTX treatment.

FIG. 11. (A) A direct-repeat recombination assay, as in FIG. 5A. (B) The assay in (A) applied to the CHO-13/14 and CGS-5/6 meganucleases. Light bars indicate the percentage of GFP+ cells when cells are transfected with the reporter plasmid alone (−endonuclease). Dark bars indicate the percentage of GFP+ cells when cells are co-transfected with a reporter plasmid and the corresponding meganuclease gene (+endonuclease). The assay was performed in triplicate and standard deviation is shown. (C) Alignment of sequences obtained from CHO cells transfected with mRNA encoding the CGS-5/6 meganuclease. The top sequence is from a wild-type (WT) CHO cell with the recognition sequence for CGS-5/6 underlined. Dashes indicate deleted bases. Bases that are italicized and in bold are point mutations or insertions relative to the wild-type sequence. Note that the mutations observed in at least clones 6d4, 6g5, 3b7, 3d11, 3e5, 6f10, 6hH8, 6d10, 6d7, 3g8, and 3a9 are expected to knockout GS gene function.

2.1.1 Gene Targeting to the CHO DHFR Locus

The CHO DHFR locus is diagrammed in FIG. 2A. The locus comprises the DHFR gene coding sequence in 6 exons spanning ˜24,500 base pairs. The Msh3 gene is located immediately upstream of DHFR and is transcribed divergently from the same promoter as DHFR. A hypothetical gene, 2BE2121, can be found ˜65,000 base pairs downstream of the DHFR coding sequence. Thus, there is a ˜65,000 base pair region downstream of the DHFR gene that does not harbor any known genes and is a suitable location for targeting the insertion of sequences of interest. Target sites for insertion of a sequence of interest generally should not be selected which are <1,000 base pairs, and preferably not <5,000 base pairs from either the DHFR or 2BE2121 genes. This limits the window of preferred target sites to the region 1,000-60,000 base pairs, or 5,000-60,000 base pairs downstream of the DHFR coding sequence. The sequence of this region is provided as SEQ ID NO: 2.

The human and mouse DHFR loci have an organization similar to CHO locus. In both cases, the Msh3 gene is immediately upstream of DHFR but there is a large area devoid of coding sequences downstream of DHFR. In humans, the ANKRD34B gene is ˜55,000 base pairs downstream of DHFR while the ANKRD34B gene is ˜37,000 base pairs downstream of DHFR in mouse. Therefore, the genomic region downstream of DHFR is an appropriate location to insert genes of interest in CHO, human, and mouse cells and cell lines. Further, gene expression cassettes inserted into this region will be expressed at a high level, resistant to gene silencing, and capable of being amplified by treatment with MTX. Methods for amplifying the CHO cell DHFR locus are known in the art (see, e.g., Kellems, ed., Gene amplification in mammalian cells: a comprehensive guide. Marcel Dekker, New York, 1993) and typically involve gradually increasing the concentration of MTX in the growth media from 0 to as high as 0.8 mM over a period of several weeks.

2.1.2 Gene Targeting to the GS Locus

The CHO, human, and mouse glutamine synthetase (also known as “glutamate-ammonia ligase” or “GluL”) loci share a common organization (FIG. 2B). The TEDDM1 gene is immediately upstream of GS in all species (5,000 bp upstream in the case of human, ˜7,000 bp upstream in the case of mouse and CHO). The closest downstream gene, however, is ˜46,000 away in the case of human and ˜117,000 bp away in the case of mouse and CHO. Therefore, we predict that the chromosomal region 1,000-41,000 bp, or 5,000-41,000 bp downstream of GS in human cells and 1,000-100,000 bp, or 5,000-100,000 bp downstream of GS in mouse and CHO cells are appropriate locations to target the insertion of sequences of interest. Because DNA sites distal to the GS coding sequence are more likely to be susceptible to gene silencing, the chromosomal region 5,000-60,000 bp downstream of GS is a preferred location to target the insertion of a sequence of interest even in mouse or CHO cells. The sequence of this region from the CHO genome is provided as SEQ ID NO: 3. Gene expression cassettes inserted into this region will be expressed at a high level, resistant to gene silencing, and capable of being amplified by treatment with MSX. Less-preferred regions include the chromosomal region between the TEDDM1 and GS genes or the region <10,000 bp downstream of TEDDM1 (see FIG. 2B). Methods for amplifying the GS locus are known in the art (Bebbington et al. (1992), Biotechnology (N Y). 10(2):169-75).

2.2 Engineered Endonucleases for Gene Targeting

A sequence of interest may be inserted into an amplifiable locus using an engineered site-specific endonuclease. Methods for generating site-specific endonucleases which can target DNA breaks to pre-determined loci in a genome are known in the art. These include zinc-finger nucleases (Le Provost et al. (2010), Trends Biotechnol. 28(3):134-41), TAL-effector nucleases (Li et al. (2011), Nucleic Acids Res. 39(1):359-72), and engineered meganucleases (WO 2007/047859; WO 2007/049156; WO 2009/059195). In one embodiment, the invention provides engineered meganucleases derived from I-CreI that can be used to target the insertion of a gene of interest to an amplifiable locus. Methods to produce such engineered meganucleases are known in the art (see, e.g., WO 2007/047859; WO 2007/049156; WO 2009/059195). In preferred embodiments, a “single-chain” meganuclease is used to target gene insertion to an amplifiable region of the genome. Methods for producing such “single-chain” meganucleases are known in the art (see, e.g., WO 2009/059195 and WO 2009/095742). In some embodiments, the engineered nuclease is fused to a nuclear localization signal (NLS) to facilitate nuclear uptake. Examples of nuclear localization signals include the SV40 NLS (amino acid sequence MAPKKKRKV (SEQ ID NO: 36)) which can be fused to the C- or, preferably, the N-terminus of the protein. In addition, an engineered nuclease may be tagged with a peptide epitope (e.g., an HA, FLAG, or Myc epitope) to monitor expression levels or localization or to facilitate purification.

2.3 Engineered Cell Lines with Sequences of Interest Targeted to Amplifiable Loci

In some embodiments, the invention provides methods for using engineered nucleases to target the insertion of transgenes into amplifiable loci in cultured mammalian cells. This method has two primary components: (1) an engineered nuclease; and (2) a donor DNA molecule comprising a sequence of interest. The method comprises contacting the DNA of the cell with the engineered nuclease to create a double strand DNA break in an endogenous recognition sequence in an amplifiable locus followed by the insertion of the donor DNA molecule at the site of the DNA break. Such insertion of the donor DNA is facilitated by the cellular DNA-repair machinery and can occur by either the non-homologous end-joining pathway or by homologous recombination (FIG. 1).

The engineered nuclease can be delivered to the cell in the form protein or, preferably, as a nucleic acid encoding the engineered nuclease. Such nucleic acid can be DNA (e.g., circular or linearized plasmid DNA or PCR products) or RNA. For embodiments in which the engineered nuclease coding sequence is delivered in DNA form, it should be operably linked to a promoter to facilitate transcription of the engineered nuclease gene. Mammalian promoters suitable for the invention include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc Natl Acad Sci U S A. 81(3):659-63) or the SV40 early promoter (Benoist and Chambon (1981), Nature. 290(5804):304-10) as well as inducible promoters such as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45).

In some embodiments, mRNA encoding the engineered nuclease is delivered to the cell because this reduces the likelihood that the gene encoding the engineered nuclease will integrate into the genome of the cell. Such mRNA encoding an engineered nuclease can be produced using methods known in the art such as in vitro transcription. In some embodiments, the mRNA is capped using 7-methyl-guanosine. In some embodiments, the mRNA may be polyadenylated.

Purified engineered nuclease proteins can be delivered into cells to cleave genomic DNA, which allows for homologous recombination or non-homologous end-joining at the cleavage site with a sequence of interest, by a variety of different mechanisms known in the art. For example, the recombinant nuclease protein can be introduced into a cell by techniques including, but not limited to, microinjection or liposome transfections (see, e.g., Lipofectamine™, Invitrogen Corp., Carlsbad, Calif.). The liposome formulation can be used to facilitate lipid bilayer fusion with a target cell, thereby allowing the contents of the liposome or proteins associated with its surface to be brought into the cell. Alternatively, the enzyme can be fused to an appropriate uptake peptide such as that from the HIV TAT protein to direct cellular uptake (see, e.g., Hudecz et al. (2005), Med. Res. Rev. 25: 679-736).

Alternatively, gene sequences encoding the engineered nuclease protein are inserted into a vector and transfected into a eukaryotic cell using techniques known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley 1999). The sequence of interest can be introduced in the same vector, a different vector, or by other means known in the art. Non-limiting examples of vectors for DNA transfection include virus vectors, plasmids, cosmids, and YAC vectors. Transfection of DNA sequences can be accomplished by a variety of methods known to those of skill in the art. For instance, liposomes and immunoliposomes are used to deliver DNA sequences to cells (see, e.g., Lasic et al. (1995), Science 267: 1275-76). In addition, viruses can be utilized to introduce vectors into cells (see, e.g., U.S. Pat. No. 7,037,492). Alternatively, transfection strategies can be utilized such that the vectors are introduced as naked DNA (see, e.g., Rui et al. (2002), Life Sci. 71(15): 1771-8).

General methods for delivering nucleic acids into cells include: (1) chemical methods (Graham et al. (1973), Virology 54(2):536-539; Zatloukal et al. (1992), Ann. N.Y. Acad. Sci., 660:136-153; (2) physical methods such as microinjection (Capecchi (1980), Cell 22(2):479-488, electroporation (Wong et al. (1982), Biochim. Biophys. Res. Commun. 107(2):584-587; Fromm et al. (1985), Proc. Nat'l Acad. Sci. USA 82(17):5824-5828; U.S. Pat. No. 5,384,253) and ballistic injection (Johnston et al. (1994), Methods Cell. Biol. 43(A): 353-365; Fynan et al. (1993), Proc. Nat'l Acad. Sci. USA 90(24): 11478-11482); (3) viral vectors (Clapp (1993), Clin. Perinatol. 20(1): 155-168; Lu et al. (1993), J. Exp. Med. 178(6):2089-2096; Eglitis et al. (1988), Avd. Exp. Med. Biol. 241:19-27; Eglitis et al. (1988), Biotechniques 6(7):608-614); and (4) receptor-mediated mechanisms (Curiel et al. (1991), Proc. Nat'l Acad. Sci. USA 88(19):8850-8854; Curiel et al. (1992), Hum. Gen. Ther.

3(2):147-154; Wagner et al. (1992), Proc. Nat'l Acad. Sci. USA 89 (13):6099-6103). In some preferred embodiments, 7-methyl-guanosine capped mRNA encoding the engineered nuclease is delivered to cells using electroporation.

The donor DNA molecule comprises a gene of interest operably linked to a promoter. In many cases, a donor molecule may comprise multiple genes operably linked to the same or different promoters. For example, donor molecules comprising monoclonal antibody expression cassettes may comprise a gene encoding the antibody heavy chain and a second gene encoding the antibody light chain. Both genes may be under the control of different promoters or they may be under the control of the same promoter by using, for example, an internal-ribosome entry site (IRES). Donor molecules may also comprise a selectable marker gene operably linked to a promoter to facilitate the identification of transgenic cells. Such selectable markers are known in the art and include neomycin phosphotransferase (NEO), hypoxanthine phosphoribosyltransferase (HPRT), glutamine synthetase (GS), dihydrofolate reductase (DHFR), and hygromycin phosphotransferase (HYG) genes.

In some embodiments, donor DNA molecules will additionally comprise flanking sequences homologous to the target sequences in the DNA of the cell. Such homologous flanking sequences comprise >3 or, preferably, >50 or, more preferably, >200 or, most preferably, >400 base pairs of DNA that are identical or nearly identical in sequence to the chromosomal locus recognized by the engineered nuclease (FIG. 1). Such homologous DNA sequences facilitate the integration of the donor DNA sequence into the amplifiable locus by homologous recombination.

The “donor” DNA molecule can be circular (e.g., plasmid DNA) or linear (e.g., linearized plasmid or PCR products). Methods for delivering DNA molecules are known in the art, as discussed above.

In some embodiments, the engineered nuclease gene and donor DNA are carried on separate nucleic acid molecules which are co-transfected into cells or cell lines. For example, the engineered nuclease gene operably linked to a promoter can be transfected in plasmid form simultaneously with a separate donor DNA molecule in plasmid or PCR product form. In an alternative embodiment, the engineered nuclease can be delivered in mRNA form with a separate donor DNA molecule in plasmid or PCR product form. In a third embodiment, the engineered nuclease gene and donor DNA are carried on the same DNA molecule, such as a plasmid. In a fourth embodiment, cells are co-transfected with purified engineered nuclease protein and a donor DNA molecule in plasmid or PCR product form.

Following transfection with the engineered nuclease and donor DNA, cells are typically allowed to recover from transfection (24-72 hours) before being cloned using methods known in the art. Common methods for cloning a genetically engineered cell line include “limiting dilution” in which transfected cells are transferred to tissue culture plates (e.g., 48 well, 96 well plates) at a concentration of <1 cell per well and expanded into clonal populations. Other cloning strategies include robotic clone identification/isolation systems such as ClonePix™ (Genetix, Molecular Devices, Inc., Sunnyvale, CA). Clonal cell lines can then be screened to identify cell lines in which the sequence of interest is integrated into the intended target site. Cell lines can easily be screened using molecular analyses known in the art such as PCR or Southern Blot. For example, genomic DNA can be isolated from a clonal cell line and subjected to PCR amplification using a first (sense-strand) primer that anneals to a DNA sequence in the sequence of interest and a second (anti-sense strand) primer that anneals to a sequence in the amplifiable locus. If the donor DNA molecule comprises a DNA sequence homologous to the target site, it is important that the second primer is designed to anneal to a sequence in the amplifiable locus that is beyond the limits of homology carried on the donor molecule to avoid false positive results. Alternatively, cell lines can be screened for expression of the sequence of interest. For example, if the sequence of interest encodes a secreted protein such as an antibody, the growth media can be sampled from isolated clonal cell lines and assayed for the presence of antibody protein using methods known in the art such as Western Blot or Enzyme-Linked Immunosorbant Assay (ELISA). This type of functional screen can be used to identify clonal cell lines which carry at least one copy of the sequence of interest integrated into the genome. Additional molecular analyses such as PCR or Southern blot can then be used to determine which of these transgenic cell lines carry the sequence of interest targeted to the amplifiable locus of interest, as described above.

The method of the invention can be used on any culturable and transfectable cell type such as immortalized cell lines and stem cells. In preferred embodiments, the method of the invention is used to genetically modify immortalized cell lines that are commonly used for biomanufacturing. This includes:

-   -   1. Hamster cell lines such as baby hamster kidney (BHK) cells         and all variants of Chinese Hamster Ovary (CHO) cells, e.g.,         CHO-K1, CHO-S (Invitrogen Corp., Carlsbad, CA), DG44, or         Potelligent™ (Lonza Group Ltd., Basel, Switzerland). Because the         genome sequences of different hamster cell lines are very nearly         identical, an engineered meganuclease which can be used to         practice the invention in one hamster cell type (e.g., BHK         cells) can generally be used to practice the invention in         another hamster cell type (e.g., CHO-K1).     -   2. Mouse cell lines such as mouse hybridoma or mouse myeloma         (e.g., NS0) cells. Because the genome sequences of different         mouse cell lines are very nearly identical, an engineered         meganuclease which can be used to practice the invention in one         mouse cell type (e.g., mouse hybridoma cells) can generally be         used to practice the invention in another mouse cell type (e.g.,         NS0).     -   3. Human cell lines such as human embryonic kidney cells (e.g.,         HEK-293 or 293S) and human retinal cells (e.g., PER.C6). Because         the genome sequences of different human cell lines are very         nearly identical, an engineered meganuclease which can be used         to practice the invention in one human cell type (e.g., HEK-293         cells) can generally be used to practice the invention in         another human cell type (e.g., PER.C6).

2.6 Pre-Engineered Cell Lines with Engineered Target Sequences in Amplifiable Loci

In one embodiment, the invention provides cell lines which are pre-engineered to comprise a targetable “engineered target sequence” for gene insertion in an amplifiable locus in a mammalian cell line (FIG. 3). An engineered target sequence comprises a recognition sequence for an enzyme which is useful for inserting transgenic nucleic acids into chromosomal DNA sequences. Such engineered target sequences can include recognition sequences for engineered meganucleases derived from I-CreI (e.g., SEQ ID NO 37-87 from WO 2009/076292), recognition sequences for zinc-finger nucleases, recognition sequences for TAL effector nucleases (TALENs), the LoxP site (SEQ ID NO 4) which is recognized by Cre recombinase, the FRT site (SEQ ID NO: 5) which is recognized by FLP recombinase, the attB site (SEQ ID NO: 6) which is recognized by lambda recombinase, or any other DNA sequence known in the art that is recognized by a site specific endonuclease, recombinase, integrase, or transpose that is useful for targeting the insertion of nucleic acids into a genome. Thus, the invention allows one skilled in the art to use an engineered nuclease (e.g., a meganuclease, zinc-finger nuclease, or TAL effector nuclease) to insert an engineered target sequence into an amplifiable locus in a mammalian cell line. The resulting cell line comprising such an engineered target sequence at an amplifiable locus can then be contacted with the appropriate enzyme (e.g., a second engineered meganuclease, a second zinc-finger nuclease, a second TAL effector nuclease, a recombinase, an integrase, or a transposase) to target the insertion of a gene of interest into the amplifiable locus at the engineered target sequence. This two-step approach can be advantageous because the efficiency of gene insertion that can be achieved using an optimal meganuclease, zinc-finger nuclease, recombinase, integrase, or transposase might be higher than what can be achieved using the initial endonuclease (e.g., meganuclease or zinc-finger nuclease) that cleaves the endogenous target site to promote insertion of the engineered target sequence.

In an alternative embodiment, a cell line is produced by inserting an engineered target sequence into an amplifiable locus with the concomitant removal of all or a portion of the adjacent endogenous marker gene (FIG. 4). For example, an engineered meganuclease, zinc-finger nuclease, or TAL-effector nuclease can be used to remove the first two exons of both alleles of the CHO DHFR gene and replace them with an engineered target sequence for a different engineered meganuclease, ZFN, TALEN, recombinase, integrase, or transposase. The resulting cell line will be DHFR deficient and unable to grow in the absence of hypoxanthine/thymidine. Alternatively, for example, an engineered meganuclease, ZFN or TALEN can be used to remove the first exon of both alleles of the CHO GS gene and replace it with an engineered target sequence for a different engineered meganuclease, ZFN, TALEN, recombinase, integrase, or transposase (FIG. 4). The resulting cell line will be GS deficient and unable to grow in the absence of L-glutamine. Such a cell line is useful because a gene of interest can be inserted into the engineered target sequence in the pre-engineered cell line while simultaneously reconstituting the selectable gene (e.g., DHFR or GS). Thus, it is possible to select for transfectants harboring the gene of interest at the amplifiable locus using media conditions that select for DHFR+ or GS+ cells.

In an alternative embodiment, a cell line is produced in which an engineered target sequence is inserted into an amplifiable locus with disruption of the selectable gene (FIGS. 5, 6). This can be accomplished, for example, using a meganuclease which recognizes a DNA site in the coding sequence of the selectable gene. Such a meganuclease can be used to target the insertion of an engineered target sequence into the selectable gene coding sequence resulting in disruption of gene function by, for example, introducing a frameshift (FIG. 5). Alternatively, for example, an engineered target sequence can be inserted into an intron in the selectable gene sequence with an additional sequence that promotes improper processing of the selectable gene transcript (FIG. 6). Such sequences that promote improper processing include, for example, artificial splice acceptors or polyadenylation signals. Splice acceptor sequences are known in the art (Clancy (2008), “RNA Splicing: Introns, Exons and Spliceosome,” Nature Education 1:1) and typically comprise a 20-50 base pair pyrimidine-rich sequence followed by a sequence (C/T)AG(A/G). SEQ ID NO: 33 is an example of a splice acceptor sequence. Likewise, polyadenylation signals are known in the art and include, for example, the SV40 polyadenylation signal (SEQ ID NO: 34) and the BGH polyadenylation signal (SEQ ID NO: 35). In some embodiments, the resulting cell line harboring the new engineered target sequence in all alleles of the selectable gene will be deficient in the function of the gene due to mis-transcription or mis-translation and will be able to grow only under permissive conditions. For example, an engineered target sequence can be inserted into the GS gene sequence using a meganuclease resulting in a cell line that is GS−/− that can grow only in the presence of L-glutamine in the growth media. In a subsequent step, a gene of interest can be inserted into the engineered target sequence while simultaneously reconstituting the selectable gene (e.g., DHFR or GS). Thus, it is possible to select for transfectants harboring the gene of interest at the amplifiable locus using media conditions that select for DHFR+ or GS+ cells.

2.5 Transgenic Cell Lines for Biomanufacturing

In some embodiments, the invention provides transgenic cell lines suitable for the production of protein pharmaceuticals. Such transgenic cell lines comprise a population of cells in which a gene of interest, operably linked to a promoter, is inserted into the genome of the cell at an amplifiable locus wherein the gene of interest encodes a protein therapeutic. Examples of protein therapeutics include: monoclonal antibodies, antibody fragments, erythropoietin, tissue-type plasminogen activator, Factor VIII, Factor IX, insulin, colony stimulating factors, interferons (e.g., interferon-α, interferon-β, and interferon-γ), interleukins (e.g., interleukin-2), vaccines, tumor necrosis factor, and glucocerebrosidase. Protein therapeutics are also referred to as “biologics” or “biopharmaceuticals.”

To be used for biomanufacturing, a transgenic cell line of the invention should undergo: (1) adaptation to serum-free growth in suspension; and (2) amplification of the gene of interest. In some embodiments, the invention is practiced on adherent cell lines which can be adapted to growth in suspension to facilitate their maintenance in shaker-flasks or stirred-tank bioreactors as is typical of industrial biomanufacturing. Methods for adapting adherent cells to growth in suspension are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). For regulatory reasons, it is generally necessary to further adapt biomanufacturing cell lines to chemically-defined media lacking animal-derived components (i.e., “serum-free” media). Methods for preparing such media and adapting cell lines to it are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Such media can also be purchased commercially (e.g., CD-3 media for maintenance of CHO cells, available from Sigma-Aldrich, St. Louis, Mo.) and cells can be adapted to it by following the manufacturers' instructions. In some embodiments, the cell line is adapted to growth in suspension and/or serum-free media prior to being transfected with the engineered nuclease.

Lastly, methods for gene amplification are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). In general, the process involves adding an inhibitor of a selectable gene product to the growth media to select for cells that express abnormally high amounts of the gene product due to gene-duplication events. In general, the concentration of inhibitor added to the growth media is increased slowly over a period of weeks until the desired level of gene amplification is achieved. Inhibitor is then generally removed from the media prior to initiating a bioproduction run to avoid the possibility of the inhibitor contaminating the protein therapeutic formulation. For example, the CHO DHFR locus can be amplified by slowly increasing the concentration of MTX in the growth media from 0 mM to as high as 0.8 mM over a period of several weeks. The GS locus can, likewise, be amplified by slowly increasing the concentration of MSX in the media from 0 μM to as high as 100 μM over a period of several weeks. Methods for evaluating gene amplification are known in the art and include Southern Blot and quantitative real-time PCR (rtPCR). In addition, or as an alternative, expression levels of the sequence of interest, which are generally correlated to gene copy number, can be evaluated by determining the concentration of protein therapeutic in the growth media using conventional methods such as Western Blot or ELISA.

Following cell line production, adaptation, and amplification, protein therapeutics can be produced and purified using methods that are standard in the biopharmaceutical industry.

EXAMPLES

This invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below. Example 1 refers to engineered meganucleases that can be used to target the insertion of a gene of interest downstream of the DHFR gene in CHO cells. Example 2 refers to engineered meganucleases that can be used to target the insertion of an engineered target sequence into the CHO DHFR gene with concomitant removal of DHFR exons 1 and 2. Example 2 also refers to engineered meganucleases that can be used to target the insertion of an engineered target sequence into the CHO GS gene. Example 3 refers to meganucleases that can be used to target the insertion of a gene of interest downstream of the GS gene in CHO cells.

Example 1

Targeted Gene Insertion into the CHO DHFR Locus Using Engineered Meganucleases

The CHO genomic DNA sequence 10,000-55,000 base pairs downstream of the DHFR gene was searched to identify DNA sites amenable to targeting with engineered meganucleases. Two sites (SEQ ID NO: 7 and SEQ ID NO: 8) were selected which are, respectively, 35,699 and 15,898 base pairs downstream of the DHFR coding sequence (Table 2).

TABLE 2 Example Recognition Sites For Engineered Meganucleases in the CHO DHFR Locus. SEQ Location Relative ID Target Site to CHO DHFR NO: Sequences Coding Sequence 7 5′-TAAGGCCTCATAT 35,699 bp downstream GAAAATATA-3′ 8 5′-ATAGATGTCTTG 15,898 bp downstream CATACTCTAG-3′ 1. Meganucleases that Recognize SEQ ID NO: 7 and SEQ ID NO: 8

An engineered meganuclease (SEQ ID NO: 9) was produced which recognizes and cleaves SEQ ID NO: 7. This meganuclease is called “CHO-23/24”. A second engineered meganuclease (SEQ ID NO: 10) was produced which recognizes and cleaves SEQ ID NO: 8. This meganuclease is called “CHO-51/52.” Each meganuclease comprises an N-terminal nuclease-localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit.

2. Site-specific Cleavage of Plasmid DNA by Meganucleases CHO-23/24 and CHO-51/52

CHO-23/24 and CHO-51/52 were evaluated using a direct-repeat recombination assay as described previously (Gao et al. (2010), Plant J. 61(1):176-87, FIG. 7A). A defective GFP reporter cassette was generated by first cloning a 5′ 480 bp fragment of the GFP gene into Nhel/Hindlll-digested pcDNA5/FRT (Invitrogen Corp., Carlsbad, Calif.) resulting in the plasmid pGF. Next, a 3′ 480 bp fragment of the GFP gene (including a 240 bp sequence duplicated in the 5′ 480 bp fragment) was cloned into BamHI/XhoI-digested pGF. The resulting plasmid, pGFFP, consists of the 5′ two-thirds of the GFP gene followed by the 3′ two-thirds of the GFP gene, interrupted by 24 bp of the pcDNA5/FRT polylinker. To insert the meganuclease recognition sites, complementary oligonucleotides comprising the sense and anti-sense sequence of each recognition site were annealed and ligated into HindIII/BamHI-digested pGFFP.

The coding sequences of the engineered meganucleases were inserted into the mammalian expression vector pCP under the control of a constitutive (CMV) promoter. Chinese hamster ovary (CHO) cells at approximately 90% confluence were transfected in 96-well plates with 150 ng pGFFP reporter plasmid and 50 ng of meganuclease expression vector or, to determine background, 50 ng of empty pCP, using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen Corp., Carlsbad, Calif.). To determine transfection efficiency, CHO cells were transfected with 200 ng pCP GFP. Cells were washed in PBS 24 h post-transfection, trypsinized and resuspended in PBS supplemented with 3% fetal bovine serum. Cells were assayed for GFP activity using a Cell Lab Quanta SC MPL flow cytometer and the accompanying Cell Lab Quanta analysis software (Beckman Coulter, Brea, Calif.).

Results are shown in FIG. 7B. It was found that both of the engineered meganucleases were able to cleave their intended recognition sites significantly above background within the context of a plasmid-based reporter assay.

3. Site-specific Cleavage of CHO DHFR Locus by Meganucleases CHO-23/24 and CHO-51/52

To determine whether or not CHO-23/24 and CHO-51/52 are capable of cleaving their intended target sites in the CHO DHFR locus, we screened genomic DNA from CHO cells expressing either CHO-23/24 or CHO-51/52 to identify evidence of chromosome cleavage at the intended target site. This assay relies on the fact that chromosomal DNA breaks are frequently repaired by NHEJ in a manner that introduces mutations at the site of the DNA break. These mutations, typically small deletions or insertions (collectively known as “indels”) leave a telltale scar that can be detected by DNA sequencing (Gao et al. (2010), Plant J. 61(1):176-87).

CHO cells were transfected with mRNA encoding CHO-23/24 or CHO-51/52. mRNA was prepared by first producing a PCR template for an in vitro transcription reaction (SEQ ID NO: 20 and SEQ ID NO: 21). Each PCR product included a T7 promoter and 609 bp of vector sequence downstream of the meganuclease gene. The PCR product was gel purified to ensure a single template. Capped (m7G) RNA was generated using the RiboMAX T7 kit (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions and. Ribo m7G cap analog (Promega Corp., Fitchburg, Wis.) was included in the reaction and 0.5 μg of the purified meganuclease PCR product served as the DNA template. Capped RNA was purified using the SV Total RNA Isolation System (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions.

1.5×10⁶CHO-K1 cells were nucleofected with 3×10¹² copies of CHO-23/24 or CHO-51/52 mRNA (2×10⁶ copies/cell) using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The genomic DNA was then subjected to PCR to amplify the corresponding target site. In the case of cells transfected with mRNA encoding CHO-23/24, the forward and reverse PCR primers were SEQ ID NO: 16 and SEQ ID NO: 17. In the case of cells transfected with mRNA encoding CHO-51/52, the forward and reverse PCR primers were SEQ ID NO: 18 and SEQ ID NO: 19. PCR products were gel purified and cloned into pUC-19. 40 plasmids harboring PCR products derived from cells transfected with CHO-23/24 mRNA were sequenced, 13 of which were found to have mutations in the CHO-23/24 target site (FIG. 7C). 44 plasmids harboring PCR products derived from cells transfected with CHO-51/52 mRNA were sequenced, 10 of which were found to have mutations in the CHO-51/52 target site (FIG. 7D). These results indicate that CHO-23/24 and CHO-51/52 are able to cut their intended target sites downstream of the CHO DHFR gene.

4. Site-specific Integration into the CHO DHFR Locus Using an Engineered Meganuclease

To evaluate the efficiency of DNA insertion into the CHO DHFR locus using an engineered meganuclease, we prepared a donor plasmid (SEQ ID NO: 11) comprising an EcoRI restriction enzyme site flanked by DNA sequence homologous to the CHO-51/52 recognition site (FIG. 8A). Specifically, the donor plasmid of SEQ ID NO: 11 comprises a pUC-19 vector harboring a homologous recombination cassette inserted between the KpnI and HindIII restriction sites. The homologous recombination cassette comprises, in 5′to 3′-order: (i) 543 base pairs of DNA identical to the sequence immediately upstream of the CHO-51/52 cut site, including the upstream half-site of the CHO-51/52 recognition sequence and the four base pair “center sequence” separating the two half-sites comprising the CHO-51/52 recognition sequence; (ii) an EcoRI restriction enzyme site (5′-GAATTC-3′); and iii) 461 base pairs of DNA identical to the sequence immediately downstream of the CHO-51/52 cut site, including the downstream half-site of the CHO-51/52 recognition sequence and the four base pair “center sequence” separating the two half-sites comprising the CHO-51/52 recognition sequence. Note that this results in a duplication of the four base pair “center sequence” (5′-TTGC-3′) to maximize the likelihood of strand invasion by the 3′ overhangs generated by CHO-51/52 cleavage. We have discovered that donor plasmids comprising such a duplication of the center sequence are optimal substrates for gene targeting by homologous recombination.

mRNA encoding CHO-51/52 was prepared as described above. 1.5×10⁶ CHO-K1 cells were nucleofected with 3×10¹² copies of CHO 51-52 mRNA (2×10⁶ copies/cell) and 1.5 μg of the donor plasmid (SEQ ID NO: 11). Nucleofection was performed using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA was subjected to PCR using primers flanking the CHO-51/52 recognition site (SEQ ID NO: 18 and SEQ ID NO: 19). Importantly, these primers are beyond the limits of homologous sequence carried in the donor plasmid and, therefore, will amplify only the chromosomal DNA sequence and not the donor plasmid. PCR products were cloned into a pUC-19 plasmid and 48 clones were purified and digested with EcoRI (FIG. 8B). 10 plasmids yielded a restriction pattern consistent with the insertion of an EcoRI site into the CHO-51/52 recognition sequence. These data demonstrate that it is possible to use CHO-51/52 to precisely insert DNA downstream of the CHO DHFR gene at SEQ ID NO: 8.

5. Site-specific Integration of an Engineered Target Sequence into the CHO DHFR Locus

A donor plasmid (SEQ ID NO: 25) was produced comprising an FRT sequence (SEQ ID NO: 5) adjacent to a zeocin resistance gene under the control of an SV40 early promoter (FIG. 9A). This cassette was flanked by DNA sequence homologous to the CHO DHFR locus immediately upstream or downstream of the CHO-23/24 recognition sequence. CHO cells were co-transfected with this donor plasmid and mRNA encoding CHO-23/24 as described above. 72 hours post-transfection, zeocin-resistant cells were cloned by limiting dilution and expanded for approximately 3 weeks. Clonal populations were then screened by PCR using a first primer in the SV40 promoter (SEQ ID NO: 26) and a second primer in the DHFR locus (SEQ ID NO: 16) to identify cell lines carrying the FRT/Zeocin sequence downstream of the DHFR gene. One such cell line carrying the integrated FRT Insertion target sequence was subsequently co-transfected with a second donor plasmid (SEQ ID NO: 27) and a plasmid encoding Flp recombinase. SEQ ID NO: 27 comprises a GFP gene under the control of a CMV promoter, a FRT sequence, and a non-functional hygromycin resistance gene lacking an ATG start codon. Flp-stimulated recombination between FRT sites in the genome and the plasmid resulted in the incorporation of the entire plasmid sequence into the CHO genome at the site of the engineered target sequence. Such recombination restored function to the hygromycin-resistance gene by orientating it downstream of an ATG start codon integrated as part of the engineered target sequence. As such, successful integrations could be selected using hygromycin.

Hygromycin-resistant cells were cloned by limiting dilution and 24 individual clonal lines were assayed by PCR using a first primer in the hygromycin-resistance gene (SEQ ID NO: 28). All 24 clones yielded the expected PCR product (FIG. 9B), indicating that the GFP gene expression cassette was successfully inserted into the DHFR engineered target sequence in all cases. The 24 cell lines were then evaluated by flow cytometry and were found to express consistent levels of GFP (FIG. 9C).

6. Transgene Amplification

A GFP-expressing CHO line produced as described above was seeded at a density of 3×10⁵ cells/mL in 30 mL of media containing 50 nM MTX. Cells were cultured for 14 days before being re-seeded at the same density in media containing 100 nM MTX. Cells were cultured for another 14 days before being re-seeded in media containing 250 nM MTX. Following 14 days in culture, GFP expression in the treated cells was evaluated by flow cytometry and compared to GFP expression in the parental (pre-MTX) cell population (FIG. 10A). It was found that the MTX-treated cells had a distinct sub-population in which GFP expression was significantly increased. Individual high-expression cells from the MTX-treated population were then isolated using a cell sorter and 5 clones were expanded for 14 days in the absence of MTX. GFP expression in the 5 clonal cell populations was then evaluated by flow cytometry and compared with the parental (pre-MTX) cell population. It was found that the MTX-treated clones had approximately 4-6 times the GFP intensity as the pre-MTX cells. Quantitative PCR was then performed using a primer set specific for the GFP gene and it was found that the MTX-treated clones all had approximately 5-9 times as many copies of the GFP gene as the pre-MTX population. These data provide conclusive evidence that a transgene inserted downstream of the CHO DHFR gene can be amplified by treatment with MTX.

7. Stability of Gene Amplification

The five clonal cell lines expressing high levels of GFP that were produced in (6) above were then passaged for a period of 14 weeks in media with or without 250 nM MTX to evaluate the stability of gene amplification. GFP intensity was determined on a weekly basis and the quantitative PCR assay used to determine GFP gene copy number described above was repeated at the end of the 14 week evaluation period. As expected, the clones passaged in media with MTX maintained a high level of GFP expression with no clone deviating more than 20% from the GFP intensity determined in week 1. Quantitative PCR revealed that gene copy number likewise deviated by less than 20% for all clones. Surprisingly, gene amplification was equally stable in cell lines grown in media lacking MTX. Contrary to what would have been predicted based on the existing art, GFP gene expression was not reduced by more than 18% in any of the five cell lines over the 14 week evaluation period. Gene copy number determined by quantitative PCR was also stable with less than 24% deviation over time for all of the cell lines. These results indicate that a transgene amplified in the CHO DHFR locus is stable for an extended period of time, obviating the need to grow the cells in toxic selection agents that that could contaminate bioproduct formulations.

Example 2

Insertion of an Engineered Target Sequence into the CHO DHFR or GS Gene Coding Regions

As diagrammed in FIG. 4, an alternative method for targeting a sequence of interest to an amplifiable locus involves the production of a cell line in which a portion of a selectable gene is replaced by an engineered target sequence. The advantage of this approach is that the subsequent insertion of a sequence of interest can be coupled with reconstitution of the selectable gene so that cell lines harboring the properly targeted sequence of interest can be selected using the appropriate media conditions. A cell line harboring such an engineered target sequence can be produced using nuclease-induced homologous recombination. In this case, a site-specific endonuclease which cuts a recognition sequence near or within the selectable gene sequence is preferred.

1. Engineered Meganucleases that Cut within the DHFR or GS Genes

A meganuclease called “CHO-13/14” (SEQ ID NO: 12) was produced which cuts a recognition sequence in the CHO DHFR gene (SEQ ID NO: 13). The recognition sequence is in an intron between Exon 2 and Exon 3 of CHO DHFR. A meganuclease called “CGS-5/6” (SEQ ID NO: 14) was produced which cuts a recognition sequence in the CHO GS gene (SEQ ID NO: 15). Each meganuclease comprises an N-terminal nuclease-localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit.

2. Site-specific Cleavage of Plasmid DNA by Meganucleases CHO-13/14 and CGS-5/6

CHO-13/14 and CGS-5/6 were evaluated using a direct-repeat recombination assay as described in Example 1 (FIG. 7A). Both meganucleases were found to efficiently cleave their intended recognition sequences within the context of a plasmid-based reporter assay (FIG. 7B).

3. Site-specific Cleavage of the CHO GS Gene by CGS-5/6

CHO cells were transfected with mRNA encoding CGS-5/6. mRNA was prepared by first producing a PCR template for an in vitro transcription reaction (SEQ ID NO: 22). Each PCR product included a T7 promoter and 609 bp of vector sequence downstream of the meganuclease gene. The PCR product was gel purified to ensure a single template. Capped (m7G) RNA was generated using the RiboMAX T7 kit (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions and. Ribo m7G cap analog (Promega Corp., Fitchburg, Wis.) was included in the reaction and 0.5 μg of the purified meganuclease PCR product served as the DNA template. Capped RNA was purified using the SV Total RNA Isolation System (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions.

1.5×10⁶ CHO-K1 cells were nucleofected with 3×10¹² copies of CGS-5/6 using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The genomic DNA was then subjected to PCR to amplify the CGS-5/6 target site using the primers of SEQ ID NO: 23 and SEQ ID NO: 24. The PCR products were cloned into a pUC-19 plasmid and 94 plasmids harboring PCR products were digested with the BssSI restriction enzyme, which recognized and cuts the sequence 5′-CTCGTG-3′ found within the CGS-5/6 recognition sequence. 17 plasmids were found to be resistant to BssSI, suggesting that the CGS-5/6 recognition site was mutated. These 17 plasmids were sequenced to confirm the existence of indels or point mutations within the CGS-5/6 recognition sequence (FIG. 7C). These results indicate that CGS-5/6 is able to cut its intended target site within the CHO GS gene. Because the CGS-5/6 recognition sequence is within an exon in the GS coding sequence, many of the mutations introduced by CGS-5/6 are expected to frameshift the GS gene. Therefore, CGS-5/6 is useful for knocking-out CHO GS to produce GS (−/−) cell lines. Such cell lines are useful because they are amenable to GS selection and amplification for producing biomanufacturing cell lines.

Example 3 Meganucleases for Targeting Gene Insertion to the CHO GS Locus

1. Engineered Meganucleases that Cut Downstream of the CHO GS Gene

An engineered meganuclease called “CHOX-45/46” (SEQ ID NO: 29) was produced which recognizes a DNA sequence (SEQ ID NO: 30) approximately 7700 base pairs downstream of the CHO GS coding sequence. CHO cells were transfected with mRNA encoding CHOX-45/46 as described in Example 2. 72 hours post transfection, genomic DNA was extracted from the transfected cell pool and the region downstream of the CHO GS gene was PCR amplified using a pair of primers (SEQ ID NO: 31 and SEQ ID NO: 32) flanking the CHOX-45/46 recognition sequence. PCR products were then cloned and 24 cloned products were sequenced. It was found that 14 of the 24 clones PCR products (58.3%) had large mutations in the sequence consistent with meganuclease-induced genome cleavage followed by mutagenic repair by non-homologous end-joining. From these data, we conclude that the CHOX-45/46 meganuclease is able to specifically cleave a DNA site downstream of the CHO GS gene coding sequence and will likely be able to target the insertion of transgenes to this amplifiable locus in the genome.

SEQUENCE LISTING (wild-type I-CreI, Genbank Accession # P05725) SEQ ID NO: 1     1 MNTKYNKEFL LYLAGFVDGD GSIIAQIKPN QSYKFKHQLS LTFQVTQKTQ RRWFLDKLVD    61 EIGVGYVRDR GSVSDYILSE IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE QLPSAKESPD   121 KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLSEKKK SSP (Chromosomal region 5,000-55,000 base pairs downstream of CHO DHFR gene coding sequence) SEQ ID NO: 2     1 taaaactcaa gatgccagct ttgtagctag cttaggaaac aaagtagtaa aaaataataa    61 tgggtgggtg aaggtctgaa gcatttacag agttctctca agacaaagca cagaggctgg   121 tggccacata acttggcaac tgatttgggg gaacagaata caagaaagga aatttaaata   181 ctgtttttct caatgttgaa ctatatgggc atagtcacag ctgcctaacc tatagagact   241 ggaagctgga acctcggcta tctaagatag aataatcaag aaatgtcaat tatttgagaa   301 aaacatcagg aataaatagc tgctaagtta caagttggtg ctttagacat ttggagagga   361 taggatgggg gctcccagac ctggggctcc ctaataaagc tgtgctggcc tacaagttcc   421 agggatcctc cagtccatgc ctcccactgt tgggactgcg ggcgatggtt tctgacgtgg   481 gtactgaggg cctgaactgt ccacacactt aagccacacg ccttttactg agtcatctcc   541 tcatctcaga acattttcct ttaatctttc ttaatgaaaa ggtcgcattt cttccgaggg   601 ctagcctcct gttactctct atacatgtca cataaaacta catgaaaact ttgaaggcac   661 tatatgtcca tactcagatg aaaagccatt agctgtggtc atacaaaacc ccacagacca   721 actgttggga aacatcagac ttttttcctg cagcgcctgc cctgatcttc cacagagaat   781 tcagtctcac tttttccagg atgacttctg aactatcacc gtaagatgag aatttgaaac   841 aaagatgtaa gtaatgaact tcatgtgttc tgaacacaca gcttagtgca ttgaaattac   901 gtaacacccg cttccttata agccatttct caaaatgttc ccattacacc tgcatcgggg   961 atgggtccca gaatcttcct tttaaataaa caccccagag gattctgaag ctagaacacc  1021 aaggactgac agagagaagc atgcctgtgg gcgactccag acacctggga gctgcctgct  1081 ttcttgctac tgatttagaa ggcatttgcc cccgaatggg gctgggggac tgtcactatt  1141 tctcattctc gggactttga aaggaagcaa aacagaaaac catgcaaagt ataagccacc  1201 atggaataat ggcagacgat ccggttgtgc agattagatt ttacatattg ctgattttga  1261 agctaaagac ctttcacttc ttaaatatat aataaaattc atacaagagt attttgtgta  1321 ggtaactcag tcagatacaa ggtaagcaaa gtaaatgata ggtgcccctt aacaaaatgc  1381 attctcatag ttcatttatc aattatagaa atggtggact ggagggaagg cttgaggtca  1441 ggagaatgtg ctgctcttcc agacagcccg ggttcttttc cccagcaatc tgggactcac  1501 gtctgcctgt agctccaggc ccaggggatc tggcaccttc ttctggcctc tgcaggcacc  1561 catacacaca tggcatacac acacatacac aaattctaaa attaaatagt aggttgtagg  1621 cctacacaaa aacatgcata cattaactaa ataattaata gttaataaat aaaaatcaac  1681 caaacacata cactgattaa gtaacatgac tctgtaaggt caaaggcggc tgaccagctg  1741 tgggaagggt taaataataa caatcacctt tgaaagactg gacctggtga ttaaggatgt  1801 tccagctgtg tcgtggatga gaaatcaaat gcataattga atgagtgcca ggaatagaac  1861 tggagacttt ctggtgagaa tgcttttact ggcagtagag tccctgtcta aacaggagag  1921 agacctgcag tagccctgtg gcggccctgc agtggccctg tgatggctct gcagttgtac  1981 tcttcctgag ataggagaca cactagagag tgtttctaat gagcagctcc tgtactttct  2041 gttcccctgg agaccgcacg tgtttctccg ataatacatt gacatttctg ttaaaccatt  2101 ttcttcttgg aacaaaaatg gagaacaaat cagattggtg tgtggtcttt taaataactt  2161 ggtacttaat aacacaaaac aaaattatca gaggctggat tttaggtgct ctcagcatct  2221 gccacccctg agccatcagt caggtcttgg aggaacaatc tccaaggaga aaacagttct  2281 gtcctcagaa aagctggagg aatatgagat tttctacagc actcatagca aaatcattta  2341 cggaagggat cctgagtaag atggcctctt cttcatcaca tggtcatagt ctgcttcaat  2401 ggggagaata gttcaatcta gcatcgagaa atcgaaggtt cccttttgac tggcaatgcc  2461 ccatagatag atagatatag attatgtata tattgtgtaa aacacacgta tgtatatata  2521 atacacatac atgtatgtgt atacatacat acatacatac atacatacat acatacatac  2581 atacatagat acgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt  2641 ttgagactga gtttctctac tatgtagctc tggctgtcct gaaagttgct aagtagacca  2701 gactggccag accagatcca ccctcctctg cctcctaagt gctgagatta aaggcctgca  2761 cccaccccca cccagcccat cttatatttt gcttcatttc aaagtaagct ctatgcatca  2821 tttattcctg catattatta gccatggttc agtcttgttt gtgttttgga atatttactt  2881 aacaaaactt gaaaaacatt tttcaagatt tgtttgtttt taagatttat ttatttatta  2941 tgtataataa taaatattat tatgaaaaac ggtgttctgc ctgcagggca gaagagggca  3001 ccagattgaa ttacagatgg ttgtgagcca ccatgtggtt gctgggactt gaactcagga  3061 cctctggaag agcagccagt acttttaact gctgagccat ctccccaggc ccaaaataca  3121 catcttaagt gtattgccac aagcatacat cttcatggcc caatcttctg tccatcactt  3181 cagacagctc tccttctttc cctggccagt cacaacaccc tcagctatca ggaaaggccc  3241 tatgggggtt gttttgtttt cccactccag ttcccttgcc tgctctgacc tcatgagtag  3301 actcatacag gatgtgctca cttcacttgg gatgatttct ttttcaccca ttgttgctct  3361 gcccagaatt tgttcctttt tattgtctta gtgttaatca actatcaaag ccagcaacaa  3421 aaaatagtag ggaaactttt ttgatagggt aaacctgatt gattgcaggc tttggttgcc  3481 ttgtttggtc tatccccttg agagtccctt acaatgtgag ttagttagtg gctgctaact  3541 agttgaatct caacttcctt tttctttaat gtgggtattt gtaaggaata gcccccttaa  3601 atctagattc tgttctcaaa tcaagcaagc tcaaggctgt aagcatggat tcaccaactt  3661 tcctgctcaa ggaatttaaa tgtctggtct ccatcatatt actttaatag taatagttta  3721 ttatacacat gtgccagctg tatatccctt ttcttcttga tggacctatg aactctgttg  3781 aggtgagatt tgaacccctt agaaggtgct agagaagagg tacctgatgg tcaaggcaag  3841 gctgatactt attcatgggt cccacatctg ctaatgtaag caataacaga taatatgctt  3901 tgtgtttaga cccacagtgg ttgcatgtac actaagtatg tatcatcatt gtcttatcgt  3961 tcctttagaa tacagctaat aattatgacc gctattctca tagcatttat attatatgag  4021 cattgtaaat tattttgaaa tgctttaaga tatacttgag aactatgcat atcatgcgta  4081 tgttgttcta ccagctggga ccttgaaatg agatcccttg aggccagcat aaagagaaag  4141 ttttcatctc aaacaaacaa aagatacact tgataataga tgagggataa atgtcatact  4201 ttttatatag tgattgagaa tctacagatt tgggtatcct ggtcacttag gagaccaagg  4261 gaggactatt agctctagag ctatgaactt tatctccaga ttccaaagcc aatacaaact  4321 ctagccaagt tggggtgctg ttacctgtat ccctctgtca aattccaagt gttttcacca  4381 cctttactgt atctttccaa ctgttctctt ttataaccac acatagttca tggtctttcc  4441 ttctctcact tgactgtgga gtaacctaac ttgcgtgttt ccagttttcg atctcttcct  4501 taaatctaca ctagttaacc acaaagaccc tcttttctga gctgtgtcta ttctatcact  4561 gtcaccattc cttaatgctc tcccagatgc agccaaactt cactttgggc ttgagagtct  4621 tctccaggtg acagtgacta atgtctccag attgagcatc taccatctac cctgtgtatt  4681 acacatgaat agccttagct tttcagcaat agacagatag atccatagtt agccatgtca  4741 acacccttct tcatgctgtt ctcacagtaa taagtcctaa ttcctgtttt ctcccatcta  4801 aactcaaccc tgtcctaaat accttactca aatcctaatt gtatctcttc cacaaacatt  4861 tcccccttct ctccattaca aggtggaaac tcagagatcc aggtgtcttg catgttgttg  4921 attctgtcct caacaaggaa ttccccaggt tcctgcacga aggaaagcat ggaggaccat  4981 acttgaggct actggtgtag tgggaagaca ggcccaaacc atgtcacaga aacccatcac  5041 cagaaagttg ggggaggcag cccagttgtg gagcaggaga aggagaaaac aggcttgggg  5101 aactgctagc tatgctttgt cacagtcaca agaaaaaagg gccctagcct ggcctacata  5161 ttctacaact tcctgaatct ttgctctgaa atgaagaggt ttggatggct gtctgggaat  5221 tcatcttgct tgcagtgaag ctccttgggg tatttgaaac caggaagttt gaaggagttg  5281 atgctaattg ttttctaaag tgtgtgagga gtactggcag agttcaggcc ttgtgaggaa  5341 agaatcctat atctagtctg cactcctggg cacatgagac attcagctat ctcccttata  5401 aagcatagaa agtactcttg tacttgacac agaaataatt tcagtatgta gagcattaaa  5461 aaaaagtatg aatgacttag agagatggct catcagttaa aagcacatac tgctcttcca  5521 gaggtcctga gttcaattcc caacaaccac aaaaactcac acatatgcat gtgattaaaa  5581 ataaaatctc tctctctctc tctctctgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgag  5641 tgtgtgtgtg tgtgtgtgag tgtgtgagtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg  5701 tgtgtgtgtg tgtgtgtgtg tgtgtgatgg tgggcttgtg tttgcaagcc cagcactagg  5761 gagttaaggc ctcactcaca gtgccaggcc agtctaggtt acagtgagtt ctagacagcc  5821 caagctacag agtaaggtac tgacaaagaa agaaagaaag aaaaaaagaa agaaagaaag  5881 aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag aaagaaagga gagaggtgag  5941 agggagggaa ggaactggaa gggggaagga gggaaagaaa agaaaaagaa acaaccaaag  6001 gaacaaacca ctgtatgcca ttatacatta gctttgggct ttacaggtta tacactctat  6061 attgtcatag ccaatgtctc aatattccat aagaggtgtc tagttgtggg tatgttcttt  6121 cttagtcctt ttatttagac tacatgacct gtttttgcct aataggccat tagtaatact  6181 gacttctcca catgctgccc tcaaaactta ctcctggaag atctttattt aagctatgaa  6241 cgaaaatctt aaccctgtga cctgccaccc agaatgcctc tgggaacaac ctcaggcaac  6301 ctatcaagcc gcttttccaa catttggggc aacagggatt aaaattatga ttgttgtctg  6361 cctgctgagt tcaaactcac agagggacca gaagctgact cactgatatc aagcagttct  6421 aaattttcag tttaaaactc taattattaa acaggggatg tcctcagacc agcactcaag  6481 agaaggagat aggcagagct ctatgagttg agttataggc cagcctggtt ttcatagtga  6541 gttttagctc tccagagagt taccagcaag accctgtcac aaacaaataa aaacaaacaa  6601 acaattaggg gatatacata taactaaatg ataaagcctt acctagcaca ttcaagtccc  6661 caggttcaat tgctagccct gggtggggat ttggacaaat ttaaaaagac cttttttgta  6721 tcacacataa atatgactgc actggttgtt gttttccatg gaaacagaat caatgtggca  6781 tgtattttac ggcattagct catatagttg tgcaggctgg caagtgtgga atgtataggg  6841 caggccagga atcagaaatt gatacaaaat tcaggaaaga cctctgggtg caatggtgca  6901 cacctttaat tcaagcactt gaaaggcaga ggcaggtgat ctttgtgagt tccaggccag  6961 cctggtctac atagtgaatt ccgggacagc cagggcttca tagaaagaac ctgtctcaaa  7021 acacacaaac aatcagaggg aagggcttat tttgtttttg agacagggtc ttctatgtag  7081 cccaggctgg cctcaaactc atgctcttga tatgcccacc tcacaagtgc atgttaagat  7141 tacaggtgcc tgacacacac cacttttgtg aagtgctgaa gagtaagccc agggcttcat  7201 ggacgctggg caagcactgt gccagctgag ccacactccc cagtgtgcac gatactttgc  7261 aaagatagat ccatatggat gctgtgcttc tatctaaaca gaatgacaac cacactctgg  7321 caggttctgg ttcataactg agtcttattg gtcacctcct tctccatttt tcgctggtat  7381 ttctcaagga gagaccacaa atgagaagtg aagcctaact tttaatgcgg tctctcctat  7441 gtcacctaaa ttctagctca aacagggttt ctggctctta ccttttcctc gggtttctgg  7501 atacttgaag tgttaacggg catttctctt aaagaccaaa tctggccaga ttcaaatggc  7561 tggccttcaa ctcggcaaac taggaacaat aatgtccgct gcatgtggct tgtagcactc  7621 tgtttctatt catggacttg tgagtgattt ctgggaaaca cgaattataa gataagtcct  7681 tttcagtgga cttcacaagt tcaccctcag gtagtatact gtcaggtaga aacgtctttc  7741 agagaagcga gaggtgacaa gccctctggg ctggccattg tccctgctgg cattgaacag  7801 cctgttcagc acatgaaagc atcgcctgat gctcccaaag ctggagcact ggcagccccc  7861 tgcagtcagg tgtgtagggt gggttagcag gggtgcttag gcgggttttg tagttacctt  7921 ttcaacacaa atgcaaaagc cagagagaga gagagagaga gagagagaga gagagagaga  7981 gagagggaga gagagagaga gagagagaga gagagagaga gagagagaga gcaggaaagc  8041 atccaggctt tgaagcaagc cagccttcag ctctgtcctt gagccattct gagtggaatg  8101 gagtaattgt ctgcttggag aactgaagaa tagcacatgg caaagaacaa tttgtacctg  8161 gaatatattc attagcttgc atgtcaaaag gccacatgca gatagaaacc attatcttgg  8221 cattctttaa aaccttgcag ccttgagact tgaggtgcag aaacccacat gcccatgtga  8281 ctgactacct gtcgatctct ccagccctgc ctggctaaca gggacaatat agggggatgg  8341 tgggagggga cagcttagac tcctgtggac ttggattgaa agaagaacag ggaagacagg  8401 ggactgtgca aataagcact ctattaggac ctatttttgg tgtcttggga ccctcctact  8461 ggtttagctt aaattgagag gggatttggt ttgcctcact agctgtttct tcccactcaa  8521 ttcacaatta cagctttctt cattgtcatt aaaatacatt aaatgtgtac ttgttggggt  8581 aaggctttct gttgaaatct gcataaagac aatgtccaca gcccccagtc agtggaaaga  8641 gcagtaggac cagaaggcat gtgtttccat cccgagtcta tattggaatg tttgttaaaa  8701 cctgcacttg taagagacaa acactagaac catcagcttg caggtctaca ggccagtgtt  8761 gccagtgcag ataatgccca aactggaacc taaagatgaa ggcctttggg agctgaggtg  8821 gaagagtcag ctgtgatctc ccagatgtcc tcctcatgcc ccattgccac tctagcctcc  8881 cacctccaag cacatttggg atccaactgc taacccctgg tgttcttttc ttagttgaaa  8941 ttctcaggga ataacctaag agtctctgtc actcagtcta tggcatccta tgataacagc  9001 caaggctaaa tagccatcat tgttcttttt ccagatgctc agcaatgagg atgcagaggt  9061 gaacaaaggt ggttcagggc tgccctgatg atgaatttga caagccagaa tctaacaaga  9121 tcagtcggta aacagaatcc tccttcctat ccagagatgt tggcttgttc tgtcactgga  9181 tgggcatcat ttactataag tcatacaggc accagacact cagagataaa taacatgaag  9241 tttccagtct tatgcagtcc tgtctagttg acttgccagt attctcaagg aagttccacc  9301 ccagcccctg gcatccatag accaaggact ctggaatgtt ctgggaaagc tccacctgag  9361 ctcctagcac ccatatatcc aaagagtctg gaacgttatg gtggaagccc cacctctctc  9421 tccccagacc tcgccccctc aaaaagtcca ccaaagactc cccacccccc acacaccccc  9481 agatgctcaa gaccacttcc atagagtatt taaactgcct cccagaaaac agaattcatt  9541 ttttcagtct ctcttcccca tgtcctctca gggtgggggg caggggtatt agtattcaag  9601 cacctatact ggcctgtcct tggggttctg acaagatatg acctcagcta cagccactaa  9661 gatcaccacc tgtgtatatc cactatgctc ccttttaaaa gggccctgtc cacctcccat  9721 tctctctgtc tctctctctg tctctgtctc tgtgtgtgtg tgtctctgtc tctctctctc  9781 tttctctctc tctctgtctc tctctctctc tccttctctg cctgactctc cctccctccc  9841 ctgctctctt ctttcctgct gcttttgtcc ctagaggcta gtctcctctc tccccttccc  9901 ccttttccca ttcactttcc cccaataaaa aactctccac ccaagctcta tcacatggca  9961 tcattctctt gctccatgat tttaaaatca caatgaggag gggagcatgg aaaaattatc 10021 caggaagact ttatccatta aacctgggtg ctttttcttt cttccttcct tcctttcttt 10081 ccttctttct ttcttccttt cttttttcct ttcttccttt cttttttcct tttttccttt 10141 ctttttgttt tgttttgttt tgagacagcg tttctctgta gctttggaga ctgccctgaa 10201 actcaatctg tagagcaggc tggccttgag ctcacagaga tccacctgcc tctgcctccc 10261 atgtgcttga attaaaggtg tgcaccacca ctgcctggct taaaactggg ctttttctaa 10321 gtcagtttga tttggattgc tgcattggca gagaggttta ttggggtgca gaaacctttc 10381 aaccagcttt tgagctaatg atagagagaa gctcaaggaa ttggagcaat gcttgactag 10441 ggatgtcaga gggaggctat ccagaggagc ttacaactga ggtaaactta aaagttaggg 10501 agtttgtcaa cttcaaccca cagaatagag cagagccagg aggagctgag gcttctgagt 10561 gttatggtgg aagcatcacc ccaacccttg acatccatat gcctgaagag tctggaatgt 10621 tatggtggaa gttccaccca agcctccctt cccggtcgcc ctccaaaccc tgctacatct 10681 cagaaatccc accaaatgat gactccctcc cccagagata ttcaagacca ctcccacagg 10741 gtatttaaac tgccccccaa cccccagaaa atagatgtgt ggttttccaa tctctctttc 10801 ctatcacgtc tctggggagc tggcaggcca tttgggagca ttgtatccat taaacgactt 10861 ctcagtggag actctgaaag ccagaagagc ctagacagat agatgtcttg catactctag 10921 agactacaga tgccggccca gactattata tccagcaaaa gtttcaaaca ccatacaaag 10981 tcaaatttaa acagtatcta tctacaaatc caatattaca gaaggtgcta gtaggaaaac 11041 tccaaactaa gattaactat acctgtgaag acacaggaaa taatctcaca ctggcaaaag 11101 aagaaaaacc tctctctctc tctcctctct ctctctctct ctctctctct ctctctctct 11161 ctctctctct ctctctcaca cacacacaca cacacacaca cacacaccaa caccaatacc 11221 atgaacaaca aaataacagg aattaacaat aattgatgtg tgtgtatgtc cctgtgtgtg 11281 tgtccttgtg tgtgtctgtt tgtgtgtctg tgtatatgtt tgtcacctga ggggtggctc 11341 ttccttggtt tgtgaggttt ctacccaatc tataactccc ttttcttcat tcacttcctc 11401 atgtccttac tagtctctat tgtggattaa ggaaactgtg tggagaacag ttttcttcta 11461 gaaaagaaca ctagccatct catgtaatca aattggtgac tatcctaatt attatgagag 11521 agcttccgtc cagtaagtgc tagaagtaga tgcagagatc cacagacaag cactgagcca 11581 agctccagga gtcctgttga aaagagagag gaaggattgt aggagccaaa gagtcaagag 11641 catgacaggg aaacccacag agacagctga cctgggcttg tgggtgggag ctcatggact 11701 cttgaccaac aattagggaa cctgcatgag gccaacctag gaactctgca tgtgtgtgac 11761 agttgtatag catggtctgt ttgtgaggct tctagcagtg ggatcagggc ctgtccttgg 11821 cgcttgagct ggcttttggg aacctgttcc gcatgctgga ttaccacacc cagccttgat 11881 gctgggggaa gcacttggtc ctgcctcaac ttgatgcgcc ttgcattgtt ggattctcat 11941 gggaggactg cccctttctg aaaaagaaca aggagaagtg aataggggag gggattggga 12001 ggagaggaag gagaggaaac tgtgataggg atgtaaaata aattaaaaaa ttaattaatt 12061 aaaaaagaac acttgtactg gtagattggc taaaatgaaa caaagataaa agtacacagg 12121 aaaaagagag gagaaacctg gggagggggg ctccaaagag aggtgagggg gggatgggaa 12181 tggcagctta gtggaggaag gaagacatga cctacacgaa tcgagctgta gtttttatct 12241 ggagcatagg gtaaagatgt ttgaggagaa ggaggaacac atgcttgtaa aacatggtct 12301 tcagaaccag caacaatcat acagagtgtc cagggtccat gggcacatga aggacagacc 12361 aacacatatt taacagtaaa gtgtccatat ttggtatgaa agtgatgggt aaattgtcct 12421 gggactgtaa tttagttgta aaggacttgt ctggcatgtg ggtattcttg ggttccctcc 12481 ttagcactga aaaaaaaaaa aaacacacac acacacacac atatattcta gtgttttgta 12541 gaaaaggatt caaagaaagc catgatttct cttttgataa atccagaata atgtaataag 12601 aacacacagt ggtgtgattt cagcaatcaa gtacaggttg cttgtctgtt tgttgtatgg 12661 gatggttggg tggttgtttg cttggtttgt aagatgggtg ggtgggttgg tgggtggttg 12721 cttggttggg tagttggttg ggtgattggg tgggtgggta tttggttggg tgggtggtgg 12781 gttggttggt cgtttggttg ggtggggtgg gttttgtttt gagacaggga tttactctat 12841 atctcagttt gtctcaaact cactatgtgc acatgagtat gtgatgagat tatctaagac 12901 catagtgtct gtgttcatgg aatgtctctc tagcttagag aatttaaaaa atggccatgt 12961 agggaaaccc ctcagaaaag gagtttctat ggcctccaag aataagaatg gatcctccta 13021 gctcggagtc agcaaggaac tgaagccctt aattttatag acacaaagga atccattgtg 13081 tggctccttc ccagccaagt ctcagatgag tcacagacct gcatggcacc ttatgcagtc 13141 ttttgaggtc ccaagaatag gatgcagata agccatgcca gaatcccaac acacaaagcc 13201 ttagtgatat agtaaatatg tattgtgtct aggctgctgc atttctggtt atgctactgt 13261 gcagtaatac acaactaata cagatgtgat ggttaatatt atgtgacaac ttgagtgggg 13321 cacagaggta cagacacttg gtaaaccatt ctgggtgcac gtaaggatag ttttggatga 13381 cataaacatt tagattagta tgctgggtaa aatacattgt ccatcccaat gggcatgggc 13441 tttgtccaac tagatgacag ctggaataga aaagtctgcc tctctcatag ttctcaggcc 13501 tttgagctca gactagacag aactcacagg ttctctgagc tttccagctt gatgaatgtc 13561 catggcagtc ttcacactta acacctgaca gacttaatga tcatatgaac caattcaaat 13621 ctgaccatca ctcgggtcat tcttttgatt ctgtcacttt ggagaactaa taccgaggac 13681 ataaaatgcc atcacatcgt tattttcttc ctgtctgtga atatttttct tttttttctt 13741 ggtttttttt tttttttttt tttttttttt tttgtttttc tctgtgtagc tttggagcct 13801 atcctggcac ttgctctgga gaccaggctg accttgaact ctcagagatc cgcctgcctc 13861 tgcctcccga gtgctgggat taaaggcgtg taccaccaac gctcggcctg tctgtgaata 13921 tttaaaatga aaactttgga aatgttctga aaccagctgg tgtcagatag tcagagaact 13981 ttcgtaaggt aggtgtgggt tatagcataa tcccacacaa gaggctgaag caggaggatt 14041 ttgtgtttga gggcagctag agccacatgg tgagtccctg cctcaaaaca caaaagcaag 14101 acaaaaacaa gctccaaata agattcactg ggccctttct ttccttcctt ctcagtgagt 14161 ccacttgctt taaaatcagg tcttaaagac gcactagatg ctgaacttaa cagtaataat 14221 aaatatcttc tcttacagta cagattatgc tctataaaca ctgcactgat aaagttcagc 14281 cttaaccttt gttctgtaaa tgtttcctag tttttctact gccgtattat aagacaaatg 14341 tcagcatgaa ggcaggtttt tcagaaaaca cagcagctcc acagatggcc tctaatccat 14401 aatcattaaa gacaagactg caactttttc aactggaaat cattcaagat gtttttctga 14461 agtccctacc aggacacaag ccaccctggt tgctgtgtga catcagttag gtagactctg 14521 aactggcttc ccaagaaatt atacaaaagc aaggtgtcac ctagtattag cataacttct 14581 gataactact gtcttagctg gggtttctat tgctgtgaag agacaccatg accacagaaa 14641 ctcttataaa ggaaagcaat tattgggtcc agcttacagt tcagaggttt aatccattgt 14701 catgattgca ggaagtatgg tggcccacag gcagacatgg tgctggagaa gtagatgaga 14761 gttctatatc agattgacac acttcttcca acaaggccac acctccactc actctgagcc 14821 tatggggcca ttttcattca aaccaccaaa gctacaaggt agcttatacc ccagcttgct 14881 atttctgatg agacttagta aatagtctta aaagcccata aaatgactca aaactagttt 14941 ttttattatt attattagtt caaattagga agaagcttgc tttacatgtc aatcccttct 15001 ccctctccct catcaaaact agttttttgt tttttaggtt ttttttcaag acagggtttc 15061 tctgtgtagc tttggagcct atcctggcac tcgctctgga gaccaggctg gcctcgaact 15121 cacagagatc tgcctgcctt tgcctcccga gtgctgggat taaaggcatg caccaccaac 15181 acctggccaa aattagtttt aagtccagtt ctaggagctc caatgccctc ttttggcttc 15241 catgggaacc aggaacacta tatatatata tatatatata tatatatata tatatatata 15301 tatatattca ggcaaatatt tatgcatata aaaataaaat aaatcttttt tccttttttt 15361 tttaaagaag tgacattgtc ttggaatttt tgtggctgct ctgcccttat gtgtaactgg 15421 acactaccag catctaaaca ctggcctgaa accagccaaa gaaaaccttt gtgccaggtc 15481 ctgtgtcaaa gtattatgtt ccttttagga tatcctatat cctaaaggat ttattttact 15541 gatagcatct taacttcctt tgaaaggttg gtcttctcaa gcagtcctcg tggagctggc 15601 tcctcagcta atgccagggg acaataatga tcccctccca aaaccaaaca gaaaaccatg 15661 gcaactctgg tttccttggg cagcacctgc tttaagaatg agcaaatgac caatcagctc 15721 atgaaactaa atactctatt attactaaaa tatttttttg agacagggca tggaattcat 15781 cacatagttc aggttggcct tgaactcaga gagactcact tacctttgcc tcccacgtgc 15841 tggaattaaa ggcatgaacc accacaccaa acataacact tgaattttgg aagagtcctt 15901 cttccaatag atttgaggtt ttgaaaatgt ggcacagaaa atatgaattc aaatataatg 15961 aaaacaagag ataactttca actaagtttc tataggttct tgctaggaat cctaagcttg 16021 tctgaaactc tagagcttct gtttctagct tctgagtgtt agtattgtag gtatgtgccc 16081 tgcctcagtg tgatgttttt gataatctta aagaaatcaa agaaatttta taaaagacta 16141 gactgtgcta cacaaaaaga atattcagat gccaagaaag agttcttaga aattaagaaa 16201 tatgctacta gtataaatcc tttataaagt ggaatgacaa atctgatgaa atcttactaa 16261 aagtagaaaa acataaacat caaagacatg aataataaga aaatcatatt gtgcatatga 16321 ttaacctaaa acattaactt gcaaaaatag aatagtccca aaaagtaaac aaaataaata 16381 aatcaccaag aacatgatac aaggacaatt cctaggatga taaaacaaga atattcatta 16441 taaaaggccc tatcactaaa gcacaacaga aacagactca aaagataaat cttcattgtc 16501 actggagaga agtccatact atcatagcac tcagaaggaa ataaaaatca aaatgtcaaa 16561 aaggacctca gcctctgaaa cacaaataca aaatatgtcc cgccttcttg acacgcatta 16621 ctcttcaatt aacattttaa gaaaactata aactgttaaa gagagcttag tattttaaga 16681 aatctgtagc tatttctttt ataagcatga caactaagtt tccctgattt aaacagacct 16741 aaaaaaccgg tgaagtgagt ggagaaaggg gatacgaaga cagcatccca catgactgct 16801 cccagtaaag gcaaggtctt catccatttt atcctgaact ctgggaaatt tataaagaac 16861 agaaatgtat ttctctcagt tctggagcct cagtccagga cactaagtct aggtactaca 16921 ctctcacatg gtggaaagta gaaagcaagc tcacttgtca ctcactacct gatgcctctt 16981 tcatcaatcc cattgataag gaagagacct ggcatctcag tttcctaagg actcagctct 17041 tactaacatt agctgtcatt tctgggtcac tgtaacagaa agcctgacag aagcaaccca 17101 ggggaagaag gatgtatttt ggctcactgt ctctgaggat ttcaacttat cccagcaata 17161 aagggataaa ggcattgcag caggaatatg tgtggcagaa gctgtttatg tcacaataaa 17221 caaataaaca cacgctagcg cgcgcgcaca cacacacaca cacacacaca cacacacaca 17281 cacagagaga gagagagaga gagagagaga gagagagaga gagagggggg ggggcagaca 17341 gacagacaga gagggagaga ggcagagagg gagagagaga gagagagaga gagagagaga 17401 gagagagaga gagagagaga gagagaaatc aaaggcccac ctccatcaga ctggtcccat 17461 atcccaaatt tctagaacct cctaaaacaa caccatcaac tgagggagac atttttggat 17521 tgaaagcata atgccattac ccaggcagaa tctgcctgtc tgggggagtc acatttaagc 17581 catggtatca attgacctca tgtaatttca gaatactaca taaaactatc agatattttt 17641 catgatgaat ttctaaagct tgaaattccc tttgaataaa ggaccaacta cagaattttg 17701 ctgagtctac aattacatac atgaaaatgt aactacgaag tggccagcca caatgaaaat 17761 taaagtgttt gggtggtctg tctctattga tgctcttctt tgccctgttt ttttttaata 17821 ttgttgatgg tttgtttttc ttttaagata cttggcccca agaaaaaaaa tgacagcctt 17881 aattaatttt gtttactctc ctgacatttt aaaagacaaa tttatgaaga cctgactgtt 17941 ccatgtagta ttagaaagat gtaaaattaa gggttgctta agctgtgtag aattgaagag 18001 cacagcattt gagtgacagg gtacaattag agatcatcag ggatgtggca caaagtgtac 18061 tcaacctcac cttttcctgc ttagcagaga acagggtgcc tcggtgagat aggaaattaa 18121 tcaaatagaa gaagaaatag taattttaga aggatcaaat tttcctggtt agaatgatca 18181 aaactacaag acttgtaact aaaatatagt caaacccatt tcaactggaa tctgtgctat 18241 tcatgtatag attaactaga atctaatttt taaattttca tcttacttcc aaaaatattt 18301 gtccaaatac tctgtgaatg cattagtttc ttatgggaaa acatcatatc ttttgtacaa 18361 tgtgtttctt agcttgaggt tctctccaaa caggaccaag acgaggccag gaccatgtga 18421 tacaacccat agtcctcaag aaatagttgt cattttctta ttccaattgc atcccaaggt 18481 ctcatctcat tttgcgtgtg cctttgacac cccataccca cataaactaa ggtggtgtta 18541 ttttttgagg ccctgaaggt atcttcagga atccataagt gagccttaag ctgcatctgg 18601 atataggaat ctgaaagtgt cccttctctg catgatctct tctttcagtt tttcaagtca 18661 gtgtgccaca ggaatcagga acgataaatg gagaggggaa gtgcagttgc ttggtataga 18721 caccccagag ggctatttgc atcctgtcct tcaaaatctc tctgagcctt cctgcctaag 18781 ctgttttgag ttgggtttgt ggtaccagaa cccctgcccc cgccccattc tgactaatga 18841 gagagagaga gagagagaga gagagagaga gagagagaga gcagcagagc atagaatgaa 18901 agtaggttag aagggcaggt aaaagcactt tagacaagag caggtataag ggccttggac 18961 tccctcccca gaacacacac atgaaggtaa acgatggtta aaggatacag ataggatgtc 19021 gaagctggac gatcacttgc ttttgtgtgc ttgaagtgac aggctgtggc tttcgggttc 19081 atggggtctg ttgttgagtt cacagtctca ccatgttagc aagcatgtca ctattaagct 19141 ctatccccgc cccccttttt tgagacatgg tcttgctaac atacccagac cggcctagga 19201 agcactttgc agtctcagct cccctgagtg ctatgatcac tcgtgtgagc tacagtaccc 19261 aaaccagaat atgtgtgttg ggtgttatga gagtttacac attgctgcct tgaatgctgc 19321 tctgcttgag ttcctgtagg aagctgagct gggaacctaa gcttcctcct cccagatagc 19381 agtaaccctg cagagacctc ccaccaagac tagctaaccc ctccttcttg tgctgtactt 19441 agcaagaacc ccaaggttct gggtccttgt gctacagttc cagaagagta tgaacaatct 19501 tagcttttct gtatatgtgt ctgtgtctgt cctgtcagat caagtcccag cctcactgta 19561 tgcaacatga aaggctgtga aaactgtgca ttttgagaat gaacatcatt agtctccagt 19621 aagttcaaaa acaaatgaag gcagccactc ataagggtct ttaatgaggc aagggggcaa 19681 aagggtggtt tctgtttgtt caaagaagcc tgtcatacat tttcagaaaa tttagaaaca 19741 cgtatcatgt catttcacgt tagtatgaag tccttataat tcatttcata ttaaatgatt 19801 tcctttggtt agaagcaaaa ttatgcataa aatgtgttcc tttgtgtttg gagcaaaatt 19861 acaagttaca ttattagtta atattctagt tcttattttt cccaatctcc aagaagcaaa 19921 atattcccct aaaccctaaa gcatcaaatt atcctatcac acagtgacca gtcatcgtaa 19981 cctaaatatt aaagcatcag attatcctgt ctatggtgac cagtcattgt aacctaaata 20041 ttattgtaat gtggattaga gttaactata ccttttcatc acactataat gtaaacactc 20101 tccaaatctt tcaaagtctt gaaaacacaa tttataaata ctgtgttctg tttgttttga 20161 gacctgatcc ggttaggaat ttcaggctgt cctcaaactc atcatcttcc tgcctcactc 20221 aggtcctaag tgctgagatt aaaggtctat gctaccacag ccatacgaat gccatgtctc 20281 catcagctta tcacttctta acttttttct tttcttcttc tacatactgc tgagtaggag 20341 catcgatgac ctcagcctag taggaatggt tcccatgtga acccttaatc tgtaggaaga 20401 tgctggactt cttccattaa gactgatctc catttgaact tgacttgtct ctctcttgtg 20461 tggagctacc atcccatata taatcttctg gtttataaac agattgcttt accctcaaga 20521 tcctttgcta gcgcagcaat gtaagtttta atacaaacag taaggtctct gattggagtg 20581 tcatggtttg gttaagtgcc ctttccaagg gcccatatag ttaagggctc aaccaccaag 20641 tgatgcttgt ggataggagg cagggcctag tggacagtct ttaggtcatg gagctatgct 20701 gttgaggggg actgtggggt cctggtcttt ttcccactcc tttttaggtc ctagctatga 20761 ggtgagtggt tttgtcctat caagcacctc tgtcctgcca tggtgtaatt gattataact 20821 acaacctctg aaactaagcc agtataacct atttatctca agatgtaact tacaggtaat 20881 ggtaagataa agctaacaaa agacaaattg ttataatcca ggcaagcctg gccccatccc 20941 ttgggggcat ggcacagagt gtgtcaccca tctgtgcatg gcaagcagta ccctgactct 21001 gtatgctgat tcaaaggtcc cttaaagcaa actcctccca cttcctctct ttttctgcca 21061 tttctctgag gagggaggcc actgtctctc tgtctctctc tgtgtctctt tttctatctt 21121 cctctccctc tcttcccttt ccccaataaa ctttccacat taagttttgt ctgaaggtat 21181 ctgtttgtct ctcacccgcc ttttaggccc cacctaccat gggatctgcc aaaggtctca 21241 cctcgagctg tattcataac acaaatgaca gacaaagatc aaccctgaag actagtagga 21301 tgtagaaggc ctggagctga cctgaagaac actgctgact tcaacattgc ccatccgtca 21361 gttatgtagc attaaagtta tagtggttcc tcagaaagca gtctcctttg aaaacttctc 21421 gttttgtgtc taaatggaat taaatacctt gttcccgaat aattgtttta gttctcttga 21481 aagatcccgt atacttacta ttaagatgta tataaacctc aagctgaaag aatgacttcc 21541 cctatggcca gatcacaaga ctctccactg atgtgcccgt tgcaacctga ttagaggaag 21601 agggtcaaag ttccccaaga ttcagctgag ttcatgcaag ttttagaaaa aaaacaagat 21661 gttcctccac agttagaaag gagtggggct ggagggatga ctcactgaga aaggttattg 21721 tcgtacaagc atgaagacct gagctcgaag cctggcaccc atgtaaaaag aaaccatgca 21781 tggtagtgtg catcttcaat cccagcattg gggagacaga gaaagagaaa gggacatccc 21841 tagagcttcc tggtcagcca gccttggcaa gccagtgaac tccaggttca gtgagagacc 21901 tgtctgggga ggaaaaaggg agggagggag ggagagagag agagacacac acacacacac 21961 acacacacag agagagagag agagagagag agagagagag agagagagag agagattgag 22021 gaagatacct gatatcaacc tcacacactc atgtacccat gtatgtaggt accttcacac 22081 acacacacac acacacacac acacacacac acacacacac acacacacac acacacacac 22141 acggatggtg ttgaattcta aggctcttat ccacacatat atggagacaa atagaagaat 22201 tacagtcgtc cctgcctttg acgctactct gtttctccaa ccctgcttcc cagatatttt 22261 tcaacatcta ctcagccttg agtggttgca ctctgacccc aggacctctt tctgtgactt 22321 ccttggcctc ctgttttgtt tttctgatgc taaaaactga atctggggcc tcatgcacac 22381 aggaagatgc tataccaatg agctacaatt ttgttgccct ttttaatttt tgagatggtc 22441 tcactaaatt gttcaggatg gcccacttgt aattctcctg ccttagcttc ccaagtagct 22501 gggcttttat acagatctgt gcttccacac ctggctgagc agacactcat gatttcattt 22561 ctgctaatca ggtagttttc ttgcccctcg ctgccatttc ctacctgcct ttccttgcca 22621 actaaactgg ttcccacaag cgacaggcta tcatttctca gctcttccac aggttagctg 22681 tgcaatttgg tatgaatcat ttagcaagcc cagttctcct ctttgtaaaa cagatgattt 22741 agatgaaatt ttttcaaagt tctctttgaa ttaaaactat cactgccttg cttgctctct 22801 gactcttgga gaccatggcc tatccctgat tagtccttgg tccacagaag gatgggtggc 22861 attggatgtg ctgaacaatc aggtactttc atgtcacttg gagtcttaca gtaactgcat 22921 gtttcaaatg aatcctttct ggctctatta gtttcttttt tgtcactgtg aaaaaaacac 22981 ctgaaagaaa caaggcacgg tttgttctga ctctcggttc agaggatata gttcaccatg 23041 gaggcaggag cttctcacag ctgtaacagc catggagtca ggtggctagt tacagtcagc 23101 tggccttagc agtcagagag ccaagagagc tcagttgagg agagtccagc caggctgtag 23161 cccttaggac ctgctcccca gagatccact ttctacagta tcttctaaac agtgtcacta 23221 gatggtgacc aggtagtcaa gcacatgagc ctgagggata atatcattca aaccatagga 23281 ttagtctaga actgaaccag atcaagaacc aggttttctt ctcacataat agataccaca 23341 catcatgttc tcatatagag tgtgatctag gtattgtttc tccaaatgga gaagccaaca 23401 ctggatgact tacatagaaa gaaagagagg gaggaaacaa gcaagggagg gggaagagtg 23461 agaattattg gaacagtacc agtgcctcaa aatccttggt ggactagaga attagcctca 23521 ggaagaagcg actaggcttc ttacagcata gacatacagt tcttaccaga ggcacagcca 23581 tcatgggtgc catggggagc atgaagttca gctccatcca gccattccta gcgatttctg 23641 gcaacctctg tcctttgaga cacttcctga agatataaga gtccagggag agacatctga 23701 ttgctttgat cccaggatct tgggatggaa ttggtgttgt ctctgctcca gctccagggt 23761 caggaaggtg aaactggaaa cacaagctag cttttcttac ttagcaaaaa cccacaggtg 23821 acataaaaga cagattgaca cgagaacagc atggcagatt tatttagtca aagttttacc 23881 agacacaagc accttcagaa aggtaaagtc agagacctta ggggaatttt cttgccagaa 23941 tttttccaga agaatcaaca gccgtgtaac aataggacta gataaacaag taagactgga 24001 cctgcagcac aaatgtgaca ataggagttg gaatccccag gactcacata aagccatggg 24061 agccgaatgt aatggtcact tgtagtttca gcctcagatg ggggtgggga ttctccagaa 24121 taagcaggct agcaagacta gccatgttgc caagctctgg gttatattga gacactctgc 24181 ctcaatgagt aagtggaaga atgatggagg ccaacttcaa ccttggactt ccacatgaac 24241 acacatacac aatgcaacca tgcatccaca gtgtatgtac acacacacac acacacacac 24301 acacacacac acacacacac acgcaaatgg acaaagaaag aggtaaaacc tacaaggaat 24361 caactgaaca gaagccaact ggtctgcctg ttcagatcct ttttggcctc tctgtgtgct 24421 tccctttctc ctgggcatgg ggcaggcagg atctgtatgg ggtgagggtc ttcagagaag 24481 cgaacagcct tcctaggttt tatggctcag tttggtggag aggggatcta gtttctctta 24541 atcatctttt taaaaattta ttaatttatt ttttatattc caatcccagt tttccctccc 24601 tcctctcttc ccctccccca cctcccatct gttccttaga gagggtaaga cctcctctag 24661 gaagtctact aagtctgccc catcatctca ttgaggcagg accaaggcac ctctccaccc 24721 ctacactctg gtgtctaggc agaacaaggt atctctccat atagaatggg ctccactaag 24781 tcagtttgtg cattagtgtt agatcttgga cccacttcca gtggcctcat atattgtccc 24841 agtcacatcg ttgtcaccta tattaaggga gtctagttcg gtcttatgca ggttccccat 24901 ttgtcagact ggagtcagtg atctctcact agctctggtc agctgattct gtggtttccc 24961 catcatgatc ttgactcctt tgttcatatt gtcactcttg cctcacttca attgtactcc 25021 aggagcttgc ccattggtta gttgtggatt tctgcatctg cttccatcta tttctggaag 25081 agggttctat cttctctggg gttgtgaatt gtagactggg tatcttttgc tttatgtctg 25141 gtatatgctt atgagtgagt acatacaaca tttgtccttc tgggtctggg ttaccccact 25201 caggatgttt tttttctagt tctgtccatt tgcctgcaaa ttttagaatg tcattgtttc 25261 ttactgctga gtagtactgc attgtgtaaa tgtaccacat tttctttatc cattcttcag 25321 ttgaggggca tctaggttgt ttccaagttc tggttattac aaataatgtt cctatgaata 25381 tagttgagca aatgtccttg tggtatgaat gtgcctcctt tgggtatatg cacaaaagtg 25441 atatttcagg gtcttgaggt aggttgattc ctaattttct gagaaatcga catactaatt 25501 tccatggagg ctgtacaagt ttgcactccc accagcaatg gaggagtgtt ctctttactc 25561 cacatcctct ccaccataag ctgtcatcag tgtttttgat cttagccttt ctgatcagct 25621 taaaatggta tctcagggtt gttttgttaa tcatcttgag aaaaaggaat tctattttct 25681 gtgactggct ctgagagaga gagaagaggg aaaggtggga ggaatgtgtg ctttcaagac 25741 cttgtgttct cccttagctc aaagtactca ccatgaaaaa ccaccagcct ttggaggagc 25801 atgctcttgc agaggcaaga tcctggcttc ctcccatctt gaatttgcca aaatagcaaa 25861 gatgtttggg tgctggacag ccaaaaatga cagctgctca cttcacagct tcctcacgta 25921 tgattacaac tccactcatc atcaagcttt aattacatca tgagcaggct tatggctgag 25981 ccgttatcct cgcatccctt cgtctcatca ctgattcaca caaatcacta ggtgctccgg 26041 ttaatgaaaa catattcatc agtacagtga ctaattcatc aggccaacat ttacatggct 26101 cctctgcatg acaaaaatga atgtttagaa tgaataatga gtcaccagag gtgggggaca 26161 tcttctgagc acaggttgcc cttgtctttc ctggtactca atcccggctg aagagctgaa 26221 caaagctgag gttatttttc ccatgacagt gcattgtggt ttagagatct gtaagcggct 26281 tatcttgatt ggcagtttga ttggttctgg gatgtactaa gagacgtgcc tcatgggcat 26341 ttccagaaag aattaactga gggggaagct cctcgccccg agaatgggta ggagcatctg 26401 gtggggtaca gatgtaaagt ggtccaaggg agaagccgca tggcctgcct gccttcactc 26461 cttgctgctg agtgtgttta tcccatctat cccgttgttg cttctgttgc agttgcaatc 26521 ctgcttctcc aggccccagc gtagactgaa cagtggctgc ccagaaattc ccaattgaag 26581 cagccgaatg gtggactgag cacctctcag tcttcagtct ctctagtttg taggcaacca 26641 ttgttggacc caactcttag tagtaagcca atctactaaa tacagaaagg ccagtgagat 26701 ggctcagtat aggtgcttac caccaagctt ggtgacccga gttcaatccc caagactcat 26761 aaggaaagaa ctaactaccg agagttgttc tctgagctcc acacatgctg aaacatgggc 26821 ctccacatgt catgaacatg ttcacacaat acatatttat ctctatatat tcatttctta 26881 taatttttag aaaatttcat tttatgtata tgagtgtttt atctgtttgt atgtctgtgt 26941 accacatgca tgcctggtgc ctgaagaagt cataagaacg tatcagattc cctctaactg 27001 gagctaaaag aagattgaga ggtacctacc atctgagtgc taggaaccaa acctgtgtct 27061 tctggaagat cagtaagcat gcttaaccac tgagccatca tgccacttat ttgtaacaca 27121 tatccatcct attggttaca gtcctgactc atacagttag atagctgagg aacctagaat 27181 tcttctgctt ttttattaca aaacaaagaa ttttatctga cttacagttc tggccttagt 27241 cagggagctg cattgggaga tggcttctct actgtcagag tccagaggtg gccgtaaagt 27301 atcatatgac atgaggcaga aagtctaact tacttgagag ttaacttgga aatgtccaaa 27361 gagacagggg gctaagtccc tcttattgaa gagaccttcc atagaagtta gcctgacaga 27421 tggccttgcc tgaactgcat tgacagtctt acttggaagg cctgttttgg ttcctaagaa 27481 attcaaggat ccaccagaga agtgtgcagc cagcaagctg gactccctat cccaagcccc 27541 agctcctcct cagggacctc agcagtcctg tgtctagctt acctcagcga tggggggaaa 27601 gatgctgttt tcctgctaag agcacactat tttatattat tgttgacaca ggttggactg 27661 catgtaacag actctccaac aacacagtga agatacaagt gtgttttgct gcatttaaat 27721 gtctccccat ctgtccctgc taagacacct actgtccttc acatgtcact gaaaactcca 27781 ccccttatga gaagtcttcc ctgatgccat ctagacaagc taagagtgct ctgctctgca 27841 ctgagcagct tctcaactct ggggttatca ttgctctgca tcacaattag cacacgtggt 27901 agtggctgtg tttgtgtttt tccacaccat gagtccagac agcatccctc tcaccagcac 27961 gccataggca caagtgctca agagtagcag gacttgaaca tgtgtggttt atcatacaga 28021 cagctgctgc tcagagacca gatcaaattc aaagcaaaat agagagatga tggttcctgc 28081 catgagcgta ctgaacaagg acaaacatca ccatcataag gaactcagct gacagggagc 28141 ggtcaccaaa cttttttttc tgtaaagtga caaaaatagt taagtatttt gccctagaca 28201 tagtgggtgg tacacatgta atctcagcat ttgtcagagt gaggcagaga gttgaatgct 28261 gggctacgta gatagtctca aaaaataaat aaataagtaa ataaataaat aaataaataa 28321 aaggaagaaa taaaaaaaag aatttgttac tcaactctgc acaatggtgc aaaagaaaca 28381 ataagcatta tgtaacctag tgggtattgg ctgtttcact ttactaacag gcattgaaat 28441 ttcaattttg caaaattttc atgttccata ttacccttat ttttattctc ccctataaat 28501 ggtgactcac caatacgcaa ctggataaga ttagggtatt tttattaggg aatatgcctt 28561 acttacagag cacctaacca gccagcagga aacatagtaa agtagcgcat gccgatgaaa 28621 caaggaaaaa gaagaactac catgtgtgac ccctaaccct taaaacctct cccacatcac 28681 cctgaccatg cccattaggc gtggtcacct agccagcccc taggaggcat ggttacggtg 28741 tccccctaca ctcccctaat catttaaaga tgcaaatgca tgcttggtga tgggctaacc 28801 ttggctcatg ggctaatctt ggctcatggg ctaaccttag ctcatgggct aataatcaag 28861 gtttactaat ctctgtcaga cagccatttt ttttttgcag agaagaatcc ccatctttgg 28921 atcatttatt tattcctttt gtatatttga tgcaatttat aaccacaaga acctactatg 28981 tgactgcact gtgccagatg gcagagaaag ctaagccccg attcttgtgg catggactca 29041 cacaactcca gtacaggact gttagtgaca atctccttaa ggcataagca tactgcagtg 29101 gcagcctctg ggttaggaga caaggataca gtttatgaca cctggtatct ggaaggcatg 29161 aaacatgtca aatgctggct acacctaaga atcagcaaca tctagtctgg ccatagccta 29221 ggatgaatgt cacagggtct taggccagaa atgtatggcc gagctgtagc agggtcctct 29281 ctagggccag aattaattcc agtgtgatgg acagccaaga ccacagggat aacaaatgag 29341 cagtgccaat gacacgtgct tctccttatt attgctgcac agtgtttgtt acacatagca 29401 ttttcgcaca gtaatataat gtgcttgggt catcttgctt catatcccat cactccctcc 29461 atctccctag tgcctcccct gttacctttg cttctcagtt ttgtttctgc tttgatgtca 29521 acagcacata caagatttta tgcaatacat cacttcctga atggctctat ttggaaatca 29581 ctaaaaggta atttatggaa catttggggt ctttttgatt ttctaattta ccaaaaaatc 29641 cacctgggga aagacaatgg agttcaagga cttctaagag gggaatgtac catggtatgc 29701 tccagccagg ggaaccagtg cttcccagga gctatggctt acaaagtggg ttatcacatg 29761 aaagcaagac taaaataatc atctcaaata ttcattagat gtgggactcc taaccatctc 29821 acaatgcctc cctcggtcta cattaaataa gaaacctcca ttttgtgctt tgcgagaaaa 29881 tgactgaaga ttatacattt ggccttgaag tggaagtatt tttgaaaatc atgaatagga 29941 aaataataaa tctctcattt caacataaaa tataagggac aaggacatct actcatgctc 30001 caaggacgga cactgaattt tccatcaggt agttgcagaa cgctgtgtcg ctcaatcaaa 30061 aattcaggat gcattgctca gagtgcatta tattaaaaga tagcatcttg gaacacagga 30121 tgctcaggaa atgggaggga cattaatctg catgcagtga tcatctcctg caaagcgggc 30181 atgagagcct gatgggagac aagccatcca gatgcccata cccaggggag ctgtactggg 30241 ctgcagccct gcgccattca gccatgcacc aggctactcc ctcctcttcc agctttctcc 30301 ttctgatggc cataggatta gaagataagg gactctagtg caggtcaact gctgaccagt 30361 gtgaaaatgc acagactaca tgctggtaga tcagcacttc aaactactgt tcaccatcat 30421 ctctggaata agcactacat ttacagggtt caaacctcaa tgaatataaa caaacaaaac 30481 acacctccct tccttcactg tctcccattt ctttggttcc catctccaca tagaatttat 30541 aattaaaatt tctaagtatc tttccagaaa tacttcacac atgttataag caaatgtgct 30601 tttaaagata ctattttaaa ttatgaaaat ggttatatta gttgagataa aagaatagaa 30661 tgggaagttc cagaatttaa ggcctcatat gaaaatataa agcgctttct cttttaagtc 30721 tagggtaggt gtactagatc agcgctcagc tccataccat gaagccatcc aggagtcaga 30781 cctctctgac agccctgcca ttgtcacaga gaagtttctg tcaccagtgc tcatgctgtc 30841 agaggagcga aggagaaaag atgtgagacc tcccaagtca aagtcatcta tggataaaac 30901 cttagttgca tggcacacca gtgttaggga gtcggggaaa cacagccata gcccagcttc 30961 ctctctgttc ttgctcttat taccaccaga aagaggttgc ttagacaacc caaaccaaga 31021 cacagggctc tgtgggaggg aatcagtccc aggcttctgg cacatgctat gtcaccggaa 31081 agccccagcc ctactccgaa tccccacaag tacagcaaat atcagattat agcatttaaa 31141 ggggcactct tgccaaagag aagcaccatt ggaatagcca tgcttgagaa ctggtcctac 31201 ttactgcaga accatggata caggctccct tttgtagatg ggcttaataa atacttctat 31261 aagtgatact ctgctttgtg aaaatgacct cgtcaatatt caaagtaatc ctctggttta 31321 ggactactat gaacctgtgg ggttcattgt tcatgtggtt aaacagcaaa gagtagttag 31381 acagttgtcc tacgtcacag agggggacat atgctatgct tggttaaata gctgtcctgg 31441 tcagagggga ggcatgctat tctgcccttt ctgacagacc ctgattgcat agacatttca 31501 gtgagataaa ggaaggaagg gaagaaggag gaaagacaac attttttgct tctgttaagg 31561 tagagactat ctgtgatcca gttcagcaca gtgcctgtga gtagaagcta caggtcaggc 31621 aggagccaag gaaatgtatt gcttttctaa ttgaacaaag gacacacagc tgccatttat 31681 tttcttcatt ttgacccttc agccctgcac tgtggatatg acatcaagaa actaagcagc 31741 cattttgtga aaatgagatc taagttagta aatgtggctg aaaaagaagc cagctgcatc 31801 ctccctggat ttacgagggg gaaatgtagg catactaaat taaaacacta aaattgaccc 31861 aaagctattt tgactgatat ttaaatatag attctgctcc tggacattcc agagttcata 31921 ggacagttgc ttctgttcag aggattcctc ttcggggttg cctctccttc cttaggcctg 31981 cttgtcctgc ccaaagctgc ccaagtgcat caggccccaa accaacttct ccatcctgac 32041 gcacagcaga ctaaatatgc aactttgtgt ctcttcatcc caggacaaaa ctttcaccca 32101 gcccctgaca tctgagactc tactacaggt tatctattaa atcttttata aagaccaaga 32161 aacaaagtgt tggcatccaa actttggtaa atcatagcct tttaataaag tcaaatggac 32221 caatgtactc taacaaaaaa atatgggtct ctcatttctg aatggcagat ttcaagccct 32281 aagaaccaca atgctcacct actgggcaac actgagttac agagacccag ctcccccacc 32341 cctcaccaag ccagagaaac actctatctg aacaatcctt ggtccatgga gcaagaatta 32401 gacatagaat ttgtatctca ttgtttttta ggaaaacccc aaaggctatt atgaagtcag 32461 tttttctggg caccttttct ttcccatgac aacgagttgt gggcagtctc agcagaatac 32521 tgaagctgtg gcttggggag acagagcata tactggattg gagttcatgg gtgggtgcat 32581 ggaatcaatg ccgggcatgg gattcaagac cttatgcatg tgggtagatg ctttgttact 32641 gggataaatc ccccacctgg gatctgactt caagcacaat ctttggaagg cggcattggc 32701 tctctgctaa tttttctagc acttttattc cacttatttt ctgcttgttt gctttgggag 32761 ttttgttcgt tataagacag tcttgctgtg tatcctaggc tgatcacaaa cctgtggcag 32821 tccttttgtc agcaggccaa aattcccact ttatctctga agacagaaag tagattgagg 32881 aatatatgat aaagacactc atcaaagcca ggcatctatc tttacttttc ttaaagcatg 32941 tttttgaatg gcataaaacc atgtagacaa ggagtcttat gttgtacatg gtcctacttt 33001 gtcacttaca atataggata ctttcaataa gcttggtagc ccttgcccta ttctacttat 33061 tctgttctct cttcctcggg tcttggggag ccttcttacc aggtggggtg gcataaaggg 33121 aaaagtcaca aagctcttcc tattcctggt tcccctccta agtgtacctt gctggtggcc 33181 ttgctagcaa atgtagtata acatctgact tatctcctct cagatatggt tgttgtactt 33241 agataaattt aatctagaaa ctcaagctgt atgtctttgg ggaccagcat tacagagctc 33301 ttcccttcct gtccttacct caccttggct actgtagtaa gttaatcctg atgattcctc 33361 catgagtcct gaaactgatt agttccaaga gctggaggat gagaagggat atagcctggt 33421 gcagggacac tttccaatga ccacaagacc ttgcacaagg tacacatgga atgtgttaga 33481 ctgtctcctt tctgtcccta gcctcagttg ccccagtgtt tatcaatgtt tattaacatt 33541 gccctagcaa aaatactaca gactaggaag cttgggtaca attgaaaaga gcttctcagg 33601 gttctggata ccgggaagtg caaaggttca gcatctggac agggctgcta ttgtagtttc 33661 aaatggttct gctgcaacac ccctttgaga gaatgaacac tgcttttcac atggtggaga 33721 gtgcacagac accaacccaa ctcctgaagg ccctttctcg agggctctaa tccatcatga 33781 gggccatact ctcaggactc attacctccc caacatcccc tctctaaata gtaccacact 33841 gcatttgcat ttcaatatat cactggagat atataaatct ccagaccaca gcataccata 33901 aatcagataa ggcaggcctg ccttctatag cctttcactc agcaaaggtg tttctagccc 33961 aaagcagtct ggactctcac tctgaaacct cttgggagtg gtggccagaa atgacttccc 34021 atcatccctc tctcctgacc tggtccagca ccaggtcacc aggaaatcct ccaagtttca 34081 ttatccccac ccccaattgt ctcttgtctc tagcaaacct cttccaatac ttccttcctt 34141 ggtgggtgta gcaagccaga tgatagcctg ccaaagaagt tcacagcctc atttctggag 34201 cctatgaata tgttacattg tgtggtaaaa ggaactttgt aggtgtgatt aaattatgaa 34261 tcttgaagtg ggcagattat ccaagtgagt ccagtgaaat tgcaaaggta catcaccaac 34321 agtgaggcag gaaggccaga gggggagaag gaagcagaga ggcagaggga ggaaaagaca 34381 agccagggga ggggagtggg gggaaagaaa ggagagagag agagagagag agagagagag 34441 agagagagag agagagagag aaatatcaca cacacacaca cacacacaca cacacacaca 34501 cacacacaca cacacctgaa cctgattgtg gaggaagaaa ccactaacca aggcattcga 34561 ggcagccttt gaaagtcaca agagacaggg aaaacagatt ctctccctcg gcccttcaga 34621 atcaacacag ccccacaact gctgatttta gtcatgttaa agccaagttg gacttctgac 34681 tgccaaaact ttagacgagc aaataaatct gcactatttt aagataccaa tgtgatttgt 34741 tcatgaaaac aatcaataag gaactaataa agtagaagtg aaaattggat cacttctgaa 34801 gtttggtaat atccacagaa actggacaca tgctgacttt gtgagccata gctccacacc 34861 caggtatgcc ccctacagaa atgtgtatat aggtgggcag gagatgtcac ctgctgtgtt 34921 catagtcgca cctttagact ttcccaagcc tgagaatagc ccaaacacct accaggagca 34981 aaataaattg agatatacag acgcagtggg atactacact tctaaaagaa tgagaaaacc 35041 acgctataca ctgtatatcg tcggaacagt aacacagggg tgacaatcag gcaataggac 35101 atattctcta tggctttaga aaacataaaa atagcataac agttctgtta gtggcaatgt 35161 gttctgtttt gtgatctgta tgatgcttcg gtttgtgcaa aagctctgga cttacctttt 35221 aaatgtatgg tggtctatac cttttaaatg tatgctagat atacatgagt aaaaatgatt 35281 aaaagagatg gaggggagga gactcatgcc ttcataaaag tttgttctgt cctttctggc 35341 actgtccaag tgaatgtgtg taaacaaaga gtgacccacc ccaggtagtc caccttctta 35401 gaacctactt ctgctacaac atgtcctgtg aatgtgcacc aaatgtttac taagggatca 35461 tgccacaggg ttttgtttaa ataaagtatg tctacctagg ggtatattga ttgtctttcc 35521 ttttgagggg gggtctcaaa actacaaact agtttgtttt gagacaagta tgtagcccag 35581 gatggccttg aactcacacc ttctgtcctg cctctttccc agcactagga tggcaggtga 35641 gactatcagc ctggccccag gaaactatct ttgattgaca ttatctggtc agaaaagatc 35701 taccttttcc tccaccaggt cctccaaata catgaagagc tgaaacagtt ctgtctaccg 35761 aatttccttt tttcttgatg tttctgtgga atttaataca taaattttaa tttgcatttt 35821 tagcttttct attaagcctt aattagagta taatgaagtt atgaatttat aaaaataaaa 35881 acaaaacggt tgctcccaca atcactcagt cttgaagtga ggttctgact ttacctgaag 35941 tgggggaaga gagtgaggaa agggacctgc ggaagctgaa tctcagaccc acaagatgga 36001 tctgagatcc atccaagcga acgtggacgc agacccggag tagggacatc caggggtcat 36061 cttcatctgt cctcgctgtg cttctgcccc tttgctcctc taccagtctc agctgtcaaa 36121 gctcagtggc ctggagggga gatggggcgg ggcttaggat cgaaggcgga gcctcggaga 36181 gcatcttctg gcccccgggg cctggactgg cccgccgccc ccacctgcag cgcggcggag 36241 cgcgggcgcg tcactcccag cggaagcgcc agcctcgcgt ctggcgaggt gcgcgcttcg 36301 cggctcccgc tccagagctt cgtggcccgc ctgtgtctgc agagcagggg cgggggcccg 36361 gcggcaccga ctgggcactg agatccaagt agccactgaa tcgtagacag tcacccagct 36421 cggacagcgc gtcggggcgg gagcagatcg ggaaggtgaa ggaccactgc ggatccgaca 36481 gcgcgtccca ggtcagtcct cccgctgcac ttggggaaac tttgggatgc ggtgacggct 36541 gcgagatgag gacactgagg gtcgcgaggc cgcgtggccc ctgtgaaccc cgcgaacccg 36601 tacctgccgc gcacctgaca ccgcagctgc cagggcgggg accgaNaccc tgctgccgcg 36661 gaccactgcg ggccaccaag ggctagcggg cttcaggggc ctctcgggag cctccggctt 36721 gcccgcgccc agccgcgcgc ctccggtcct cgcgggtccc cagctccttt tggcggctcg 36781 cgcccggacc ccgcggggct gcggattccg ccgtcttcgg gcctcgtggc gctggaggag 36841 cggcccgggg gcccatggct gcagggtggc ggccccgcgg cgggagcggc gcgtgctcgg 36901 ccggtggagc gcgcgggtcg cggggttcgg ctggagcgcg tggccgcagg tgcctgtggc 36961 cgctgggcag cggaggtgag agcgcgggct ggggacgcgg agcggattgc aacctctggc 37021 tgcaggaacc agggtcgctg ggtgagcagt cctgtccccg cggcttccgg gcgtgcacat 37081 ccctggcacc cggcatccag accccatcag ctggaggcgg gctgcagagc ggcgcctgcc 37141 cgggccgagg accagtgcct cctgctctga cacgccatct caccaacgag ggcggggtgc 37201 tagattggcg ggctgcgcgg ggaccactgg ccagggcctt ctggcacaag cccttttcgt 37261 ggacagctgc ctgctctggc ttggagtgga ggagacgaaa tgagtacccc gcccccatca 37321 gcgccccaac actgtcgccc cagtcacctt cctttgccct tctccgacag caccttggac 37381 ttgctccctc ccgaattggg gaaaatctga ggaaaccagg cagggacctt ggagataccg 37441 cagcctgcat actcaacagc ctggaaatcc agtcaccttg gtacctcgct gcttcccaga 37501 cactttggag gagcaggttt gccatttcta ccccacatcc gtaccccatc ccccgtccgt 37561 ctctgctgag gaagggactc ttatgagaga agttgggatc taggtacccc ttaaggtagc 37621 cccagagtct gtggtaacta ggctcatagg taactaaaag gcatcctagc tctgtagctt 37681 tgtgagggaa acaaacctta ccaactaatt ccttcccttt ctgaatattt cttagaagac 37741 tggagaccaa cggaagccga ctgttctggc cagtctttgc accctttgct tggctctgac 37801 tctccttcct aggcagagaa acattttgct tatgacctct ggctggcctc cttccaatcg 37861 ctgcctggcc ttggactgcc catcaggact gtgatttttt ttttttttta agacctgatt 37921 aggaaaggct gcaagcctcc ggttctagaa ggctcaaact caggggtata ctcttctctg 37981 atacccatgt gctccctaat tccactgtgg caacacctct gcccttcact cccacaagaa 38041 aattggttgt caaacctctt ggggaagatg atggaggcat ccctgtggga gcagatgcag 38101 gatttggaag caaccaggaa acaaccagga gtgaggaatc ttttttaaag gctcacatga 38161 ttctggaact aagaaaagat ggagatgcca ccagtgtatg aagcttggcc tctcctcggc 38221 ccatcccacc caactcaggg aactggcata tgcaggacct gtattgggtg atgcatattt 38281 ggaacctagt acttattgaa ttcctaagca gtaaacacat tccgaatttg aaattcctca 38341 caatcatcta ctgNaatgta gatattaaac ccccaactta tgaatgatag ccccaaaatt 38401 gttaacattg agagagccca ggttccctgc cacctcttcc acaacaggac aggaactagg 38461 acaatgaata ggaccatttg agctttaggg tcatgtgccc actttacagc tccatagcca 38521 gacaactgtt ttataagaga gggcacaaag gaaaatcact gtcctgtcca aatgaataga 38581 aagctgggga tggtggcagg acaaaggcaa caggaaaaat catctccaac aaggctttcc 38641 aagcatatca gtcttatact actgccatgt tgggtaccac acaaatcagg tatctcaaac 38701 tggacgctgc ctagggaggt ctgtcatcta aaaaggcagg gagatattga gataaaatac 38761 acagaagcta gtatttaact ccaggctggc agataatagg aatgaccttg ggagggtgtg 38821 cttacctttc cttctctctt gaacaaaatg tggactggac cagatgagca ccaaggctcc 38881 accaactcta acagaccttg tgtggtgggc ttgcctgcaa acagacttga gctaggttgc 38941 tgtgcgtggg atccattcca gactcattta caaactcgta gtcagtgaaa tgtgataaac 39001 cgaacactgt agggatttct aaacaaggaa ttaaaaaact cgactccaaa tgggagagat 39061 gcaggcaaca aatcgacagt gtttatgtgc ctctgaatag ctttgatttc cttcggtagg 39121 agctgacagc tggctgacag aaagctcacc cagggagaga agagagaaaa atcaagtatg 39181 agattaggaa taatgttttc aggtaacttt ctattcccat tcggagtggg tgtctggaag 39241 ggcgagtgta gttatggctt gaattgctcc atttatccac agatattttc ttcccaaggg 39301 ctcctgattc taagatgctg ggctttgctt ctgtctccta gtttcctggt agcagggtag 39361 agagctgggg gtcccagcat tcagcctgca tattcttcct ctatcctcac tatctgctgc 39421 ctccattatt tgtggtcttt tggatctatt tggtcagaga gtcagtcttt ggtttcttgc 39481 cctggaaact gcttgttgct acttgtggtg ggggcagcat ttggaagtcc aggtgctctg 39541 cccacaaact ttcaacccat catttgtttt tcatcccttt ctcattgcca ctttgtgtgg 39601 tgcctgggac ttctgggacc tatagttcaa gggtcatata taccaatggc tcacatgaca 39661 gcactgatca ctctgccagc tctcctctct ttgcaaaact tatttcagat ttttcatttg 39721 acaatacctt tcctccagtt gtctttattc ttggcagcat atgccttgta acctttaaaa 39781 aggaaggtaa ataatttgag aaaaaatgta ccaagtcctc agtgatacat tcttactaaa 39841 gactcccagt tttaacaagg agttgggctg gagccatggc tcaacagtta agagcactac 39901 ctgctcttcc aaaggacaca aattccattc ccagaaccca catggcccct tccaaacatt 39961 gataactctc gttccagggc acctcatgcc ctttcctggc atctgagaga accagcataa 40021 acatacatgc aggtgaacat tcatacacat aaaatgaaca ttaaaaaaga aatgaaatag 40081 agaaagggtt tacataacta tttaataact aagactgcct aataatgtag ggacccataa 40141 agaaaatcta gtaagttttt acaagattcc actcaatcag accaaacatt actgttactg 40201 acagagtaaa aagtcacttc caatagtcca agaacaactt tgtttcattt ctcaggcact 40261 gtctgttttg tggcatatgt gcatggtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 40321 tgtgtacagg tgaatgctgc tcgtgtatga gcacatgcag gtgtgtgttt gcatggtgtg 40381 tagacagagt ttctgacctg cctggtccca cagctgtttg gccacaaata aacatacaga 40441 ggcttatatt aattagaaac tgtttggcct atggcttagg cttctcactg gctatctctg 40501 tcttaattat taacccataa ctactaatct atgtatttct acgtggcgtt atcttaccgg 40561 agaatacttg gtgtcctatc ttctcagcaa ctacatggcg tcttctctct gcgtcttctc 40621 cccagaattc tcctcgtctg gttgccccgc ctatactttc tacctggcta ctggccaatc 40681 agtgttttat tcatcagcca ataagagaaa catatgtgaa gaaggacatt tccctatcaa 40741 tggtgtgtgt gtgtgtgttt gtgtgtgtgt gtgtgtgtgt gtgtgtatgt gtgtacatgg 40801 gtatgtgagc acatgtgggt atatgggtgc atgtgcacct gtgtgtgtgc atggtggcta 40861 gagttgaggt tagatgtctt ccttggctgc tctccacctt ttttttattg aagctctcac 40921 tgaacttaga gctcactgat tcagctagtc tagctacccg gcctgctctg ggggtcccct 40981 gccttcactt tccatgtggc taccatatct actttacatt tatgtgggta atggggatct 41041 gaactatggg gtcctcatgc ttgcatggca agtgctttat ggactaagac atctttctag 41101 cctttacctt tttttttttt gaaagagttt ttttttgcta actgggaact caacaccaga 41161 tagctagtct actggtcact gaggcccagg gatctactat ttctgcttct cttcccaagt 41221 gctgggacta cagactgtac caccatatcc atatttcttt tagcatgagc tctggaagtc 41281 aaactcaggt cctcacgctc acaaagtaag tgttttatct accaagccat cttcccatct 41341 ctgttgtttt aaaaggcttt gaatatggga tgtgatgaag ggaggtgaaa ttctgagata 41401 aatttcttga aaagaagaat gaatcaagta ggagaacctc ctcctggtgc tgtctttcag 41461 ttccatgtcc acacagcata aacattatga ttatcattcc acagattgta attagtcttt 41521 ctctgttttg ccagtctgct cccaaaaaat gacacagaga gacttcttat taatgatgaa 41581 agctttgcct tagcttaggc ttgtttctaa ctaactcttg taacttaaat taacccattt 41641 ctattcatct acctgctgcc acgtgattca tgacttttac ctctctctca ttctgcatat 41701 cctgcttcct ctgcttctgg ctcatgatcc cgcttttctt cctctccgag tgctctgtcc 41761 ccagaagtcc cgcctaacct cttcctgcct agcaattgcc catttggctc tttactaaac 41821 caatcacagt gacacatctt cacgcagtgt aaaggagtat tctgcaacaa caggtgatga 41881 agccaacatt ccaagaggcc agggcttgcc tagggcacat agctaactta agaaaattag 41941 gatcgcattc tacatctgtc tgactctgaa ttggatctga actgtgactt gcatggaaga 42001 cccaaagacc ctgagaaagt acaatgacaa aggggctgac tctgtccaca tggtgttagc 42061 ccaggtttcc cacaggagga aaacccatcc taggcaagag aagtggtctt catcaaacac 42121 tctatgaaaa gcaaatcaga ctcaaatgtc aggatttgtg ctttacagat cgatccggta 42181 agatgaaaga acttcctgaa agtgtgtgaa ggcctaaagt cagggctgtt catggaaggc 42241 actgactaca gaatgaggtg ccagaagcct agtcagagcc tctagggaat aaagtgtcag 42301 atgatcttct aaaaaagttg aagtttcacc agtaacagaa tggccccact attaaaatgt 42361 gagcaaactc agaagtcatt gtagcatata gaagcacaga cctatggatt gctggatgga 42421 gcccaggtat tcactccatc ctgaatagcc agctggggag ctagctcagt cagttaagta 42481 tttgctatgc aaatctgagg accagacttt ggtctcctgc atccacagaa atggtgcaca 42541 cttgtaatct cagcactggg gaagcagtca gccagatcca acagctgcct agccagcgga 42601 aacagcctta tcagaaactc atgggtcctg gtgaaagata ttatctcaaa taacaaggtg 42661 ggaagctcct gaaggacact ggaggttaac ttctggataa acataggctc gccccaccac 42721 cagtgagcat gtgcctaaat ccgtacataa caatgatgta aagatggaat tcattccagt 42781 gaaaagtaag cctcctggac tctttttttt tttttgttgc tagatattct cgagacctca 42841 ggagagaagg tttgccatca tctatataac atggtactca acttccctgt agtccacaac 42901 attcctattt ctatatgatg gagaagaggc cactgcccct cccagacatc tcagtctcaa 42961 atttgttacc agttccctct cctaataagt gcttagggtt agtgttgtag agaagggctt 43021 tacatgaagt gtgtgtgtgt gtgtgtggtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 43081 tgtgtgtgtg tgtgtgtaac ctaaaggctt tccatgtttc cacactgaaa ggttcttaag 43141 actgagaaca accagataag agtccaaatt ctagaaacca tgggaaagtg taatattgaa 43201 agtcagaaca aggcatggtg gtgctcacct tgaaacccac cacttggggc agaggcagtc 43261 agatctctgt gagttcaagg cccagcctgg tctacagact gtacatagtg agttccaggg 43321 ccagaactac atagtgagat cttgtctggc caaaaatata taagtaaata aaataaatca 43381 gtacatggta acttgttctt atttcagtgt ctgtttctca agcatgactt tggcttaagg 43441 atttttccca acttgttttt gtgattgcca ctgtatcatt tctttgtgtg aagttactaa 43501 gtggtttctg tatttgatat tatgttctga cctagtttct tttcatatta aacccatttg 43561 tatatgaaaa ctgcaaagaa gtgggttttt tgttttttgg gttttttttt gtttgtttgt 43621 ttgtttgttt tttcttggtg ttctcatgtg acctttccaa tgtttgcttc cagaatagac 43681 ctgcaagttg ggatccacac tgccatctga agtcctgcac cccaagtttc aggtatgttt 43741 tgatggcaga atagcttttc tagactgtga caataggggc ataaagccac aaagcattcg 43801 ctttcctaca ggttatgcac ccactctctg agtgattggc tgtgcatcat gaatattatc 43861 aaaatggagg cagttcagtt tggagtgctg tcttttatgc gcttattcat ggcaatgcca 43921 atggaacatt cggcaacata tactactaat catgcatggt aactgaactg tgttgtgcaa 43981 ggaagacctc atatgaccta cctttgcata tgctgacctt ttctgtgaca gactcctata 44041 atactgagag tggtactgta tggaagagtg tgtgaaaatg tattgtttaa ataacagaca 44101 gatgcctcta aatacaacac ccaagcagag aaatggagca tcactggcac tttggaggcc 44161 tctgggtaac ctttccagat cacactgttt tccttcctcc accaataacc actttccctt 44221 tggatgctac tcatagttaa catctttact tttgttgttg tcccactgat gctaagaaaa 44281 ataacttcaa ctagcaagca caacactaga tgaattaaga gtgatattga ctgtgtgtgg 44341 tgagtctcag aagactagct gcctcaggat tcatgaatgc ttacaggaac cctttagcaa 44401 ggtcaggaat gagtcttagg atccatgtgg ctcatagtct ccagcctgga catggagtag 44461 cacagtgtct gagtgcccca agggaatggg cttgttcagg ctcccctccc cgtccccagt 44521 tccaacaggt ctcagatcca ggacatcaga gctgagtgaa gagcagagct aaaaggagca 44581 ccatcggagc cctagaagca gaataggggg ggacacagca cacagagaca agaactgagg 44641 ccaggctgct gtgtgctttg ggcctaagtt gacagatgaa acatggtagg gtgaccacat 44701 ggaggatgtc tgtgcacatc catcaaactg gcaggtcccc ccagcatttt ctgggagctt 44761 ggggtcctct tttccatgat cttcagcttc tgtattctat gtgcgctgtt accatttcat 44821 cttggtagag tctatccttc tgttatttct tgagagtatg tcccaattct tgcctggagg 44881 tttggctaaa tatagaattc taagcagagg gtcatttctc cttcagatat ttaaagacac 44941 tttctgtatt gtgcctcatt gccattgttg atatacctga atctaaattg atcccttggt 45001 gcgtgactta tccccacagc caagggcccc ttcccttctg gtctgtgctc tggaagtctg 45061 caggcacatg gtatgggtag ccactgtttc attcatagtt caatgctccg ataggccctt 45121 ttgatttgat aactctatcc ctttccccca ttcccgttga tgatttcttc ttttgttccc 45181 cttttgatat agtttccttg ctgatgctgt gctaaaatat tcctaccaaa aacaacctgg 45241 ggaggagagg cttcatttgg cttacaattc cagctcacag tcattgaggg aagtcagggc 45301 aggaactcaa ggcagggagc atggaggaat tgcctgctgg cttcctctct gacttactca 45361 caggttcttg taggctagct ttctgataac atctcaggac cacctgctta gcaatagtgt 45421 ggtccacagc aggtttgaac cttctgcatc agttactaat caagacattt gcccaaagac 45481 atgcccacag gccagattga tgtaggcagt tcttaaatca agtctttttt gtcaagtgac 45541 tctagactgt caagtcgaca gttgatgcta actaggacac tattctacca cttttcttgg 45601 tagaaatatt attcggatat tggagttctt ggactagttt ttctggttct ccttttcttt 45661 cttttcctgt tatttatatt tgttttatga gatagggtct ctctgtgaag ttgtcctaga 45721 ccttctggcc ctcctgctta taattcctaa gaactgatat tacaggcagg tgccatgagc 45781 ccaacgtttt ttcttttctt ttcactgcac tctgtttgag agtctcatcg tcacagtcat 45841 tcacatcttc tattgtcttg tttttctttt taaatgtgca ttggtgtttt gcctgtatgt 45901 atgtctgtgt gagggtgtca gatcttggaa ttacagttcc aaataatatt tctaccaaga 45961 aaaagtggta gttgtatcct agttggcatc aaatgtcacc ttgacagcct tgagtcacct 46021 gagaagaaag acttgattta ggagctacca tgtggttgct ggtaattgaa cccaggacct 46081 ctggaagagc acccagtgct cttaactgct gagccatctc tctggcttcc ttctattgac 46141 ttttgcaggc ttctttcttg ttcttttgca atttcatggt ctctgactgt tcttcacaga 46201 ctcttacctc atgcttaaga tgtctcttac tccttcaagg atactgagtt tttgaagttt 46261 taattctcct gactactgtc ttttccctcc tgtttgtcat tctctgtttg ccctggcctc 46321 tgtctttcat gcaggaagac ttttcatttg cttttaggtt tttattttaa ctattggttc 46381 atgactaaag ggctagatga aaaggccagt gagaaggctg gagcatatgg gtgatacttg 46441 tcaaccggga gcctcactgt ggaatgcttc agtggcatgt gaaatcctgt ggtatttgct 46501 caggcaagtg cagctgttga atgcagacca gagcagcttc cttcgaagga gtcagatgtt 46561 gctgactgtc tttctgcagc tggtcaggaa ggtgggatag acttcagctc ttttcaaaca 46621 gtggtcacca aacaaccact tgcccagaga ctttgtgctt taccattctc agagaacaga 46681 cctctggatg gccccatggt ggaagcagcg cacctgtcta tcacaggtgc tctgaaggag 46741 ttggaagaac tacccattgt ccacatttcc cacattttca catgccagct tcactctggg 46801 atctgggtga cagtggggct gacataatgg caggggttgc agtttcagac tcagagtatg 46861 tggtaggaat gctgctgtct gagggaagac tcatctgagc agtggaggct ttgcctgttc 46921 cctggcatca tttgacctgc ccctccttag aactgggaac cccagttcta aagctccctg 46981 ctttaaagat tctgtgttgg ggtaagttct tagctttctc aggctaggtc ctctgctctt 47041 gggtttccac ggcactgttg ttttccctct ggctttgtga gtggttgtct tttgaaaaac 47101 tagttagttt ggaaaatttt gggagggagt caaataagat gtatgcattt tgccatgtaa 47161 gtcctaacca agccatctgc tgtggtattt tcctgagttt ggttctgccc ctataggcag 47221 agtctgtcat cacagataat tgcattttga acttgagcat ctcccttcct tctttgtctg 47281 cctgaaaaag tctctttata aaaaaatgta atgttaattt aaaaagtatt cattattctt 47341 gtgttgtgat acatgagtat atatatgcta tgatgcatat gtgcaggttg gaggacaact 47401 ttctgtagtt ggttctctct ttctcccttc atgtaggttc tggggatcga acccaagtca 47461 tcaagcttgc acaacagcac ctttaccttc taagccttct catcagccct ttttttattg 47521 attgattggt tgattgattg attgattgat gctagggata gagcctaggg tcttttacat 47581 gctaagaaaa tgctctacca ctgaactgca ctcctagccc aacctgctaa attcttacac 47641 tgtcttcaaa aagaagctct gatgctggat tctgcaaagt ccatttttat ccctaaattc 47701 ctaaagctgt ttaaatctcg tgagtcttac tgtacagacc agctctgtgc accatcttcc 47761 acaatctcca tgacctcctc aggatgggct ggtatctctg cagctctgcc cagtgcctac 47821 caggaactta caggtgtcac caatgaattt attggtgcat gctcacttca tcttgtccct 47881 atccactttc tgctttgact ccttctggta agagacaagt gtgttaacta cttgtgctat 47941 caccacacag aaatccatat cccataatct tagtcctttt tatttactta tttttgagac 48001 agggtcacac tctgtagctc ccacactggc cttaaacact gacctcgaac tcatggtgat 48061 tctcctgcct aaacttctca aataccatga ttacaagagt gacacaccat gctgggagtc 48121 ataatcttaa gtttaaaagt gagggactgg tcagtttact gtgctaggtt gacattgtat 48181 agaaatgaac agccatgttg gtctggaaat gttcctagtt ttcatttgta caaggatatg 48241 cagtgtgtga aatagggaga gtcttaccta tgtgggtttg atcacagcaa ttaataaaat 48301 atgctctaaa taatgaaaaa agccagtaac tagtagtgtt tctgaatcct cactaaagct 48361 ttaatacatc ataaataata tatcactgca gattatgtct acatgttata catatcacat 48421 ttatagtaca atctgatctt tgtcacctac tgtaagcaca actgaaaaac aaattttctc 48481 atagctcaat attaagtcat tattatcccc ataataagta attattatcc ccataatgaa 48541 actatctatt gagggagtca gaatctgaga tagttaaata aatttaagca tgtattttta 48601 gtgtcaatgg taaaaattaa atgttcataa agcctgtatg actcctttta aagtagtttt 48661 aattttatgt gtatacatat atgcatgttt tgccttcttg tatgtctgag taccacttgt 48721 atgtctggtg cctgaggagg ccagaacgta tcagatcccc tgaaactggt attacagttt 48781 tgagctacta tgtggctgtt gggaattgaa cctggatgct ctgaaagagc agccagtgct 48841 cttaatgact aggccatctc tccattttct taaaaaaaaa tttaaaacat ttactctaag 48901 atttactttt atgtaggtgc gtgtgtgaat gtgtatggtt tatgcattgg ggtggggagg 48961 atggattagc acagtcacag aagactagag gagggtctct actattgctt tctgtcttct 49021 acccttgaga cagggtctct cactaaacct gaaactcacc tttgcagctg gggtagctgg 49081 tcagaaagat cctggaatct gtctttctcc ctggccctaa tgcttgagtt acaggcccat 49141 gtgaccatac ctgtcgtttt actggggttc tacagagtca aacccaagtc ctcacgcttg 49201 catagccagc gattttaccg actgagacat ttatctgccc caattcataa ttcttctctg 49261 cttccattaa taatcccatc tatgtcccct tcatacatat ttctgaaata gacaaaatga 49321 atacaagtta gacatcgagt ctgattaatc ttcaacttct ttgataacca ggtattgatt 49381 tctgactttt gaagatggat gaaggcacag aagtctccac tgatggaaat tccctgatca 49441 aagctgtcca tcagagccgg cttcgcctca caagactttt gctcgaaggt ggtgcttaca 49501 tcaacgagag caatgaccgt ggcgaaacac ctttaatgat tgcttgtaag accaaacaca 49561 ttgaccagca gagcgttggt agagccaaga tggttaaata ccttctagag aacagtgctg 49621 accccaacat ccaggacaaa tctgggaaaa gcgctctgat gcacgcatgc ttggaaagag 49681 cgggcccgga agtggtttcc ttgctgctca agagtggggc tgacctcagc ttgcaggacc 49741 attctggcta ctcagctctg gtgtatgcta taaatgcaga agacagagat accctcaaag 49801 tcctccttag tgcttgccag gcgaaaggaa aagaggtcat tatcataacc acagcaaagt 49861 caccctctgg gaggcatacc acccagcagt acctcaacat gcctcccgca gacatggatg 49921 agagccatcc gccagccacg ccttcagaaa ttgacatcaa gacagcctcc ttgccactct 49981 catgttcttc agagacggac c (Chromosomal region 5,000-55,000 basepairs downstream of CHO GS gene coding sequence) SEQ ID NO: 3     1 GGGCTCAGGC ATTTATCGTT CAGAGATTGA CTGAGCTGTA AAGATGGAAA GACAAACTTT    61 TTTTTTTTTT GATTGAGTCG GGGTTTCTCT ATGTAACAGC CCTGGCTGTC CAGGAACTCA   121 CTCTGTAGAC CAGGCTGGCC TTGAACTCAC AGAGATCTGC CTGCCCCTGC CTGTCGAATG   181 TTGGGATTAA AGGTGTGAGC CACCACCGCC CCGCTGACAA ACTAGACTTT TAGAATGTAT   241 TATGAGATAA GGTTTTGTTA TGTTGCCCAG GCTGGACTCA GATCTGTAGC AATCTATCTG   301 CTCCAGACTC CTGAGTGCTG GGATATACAG ACCTGAGTTA CCTGTACAGC TTTCTAATCA   361 TCCCCCGCTC CCCCAGAGAC AGGGTTTCTC TTTATTGTTT TGGAGCCTGT CCTGGCACTG   421 GCACTCACTC TGTAGACCAG GTTGGCCTCG AACTCACAGA GATCCACCTG TCTCTGCCTC   481 CTGAGTGCCG AGATTAAAGG TGTGCACCAC CAACACCCTA CTTTCTAATT CTTAAAGCAA   541 GGCTCCCAAC TCCTCCCTTG TGTGTAATCA ACAAGGTTCT TAGACCCTGT CTGCAGTGTG   601 GATTCCCACT AATAAGACAG TGGCGGCACA GTGCTGTGTG GCAGAGCAAG CGTCCATCTA   661 GTTCCTATTG TCATTCTATG ATTTGCTCTT CTGGGAGCCT TGTCATTCAG CAAGTTCCTG   721 GGCTTGTCTT GGGATTGCAA TGTGCCTCAG CTTGGCTAGT TCCTCTGCGG CAGAAGCAGT   781 GTTTGAACTC AGTGGGCACT CAGTCACTAC ATCTAACTTG TTTGAGGGCT CTCTGCATTT   841 GCTTTCCAAT TAAGGTTTAG GATGACTCCT CCCTGTGACT CTTATCATCC TGCCTATTAA   901 TGCTAAATTA GAGAGGCATT CAAGATAACT GCCGAAGATC TAATAAATAA ATGGGGTGGG   961 TGGGTAGGAC TATAAACCAG TTTATAGCAT GCAAGAAAGC TCTGAGCACC ACATTCAAAA  1021 ATAAAGTGCT GTGAGCCTGG TGGTGGTGGC TCACACCCTG ATCCCAGAAC TCAAGAAGTA  1081 GACAGAAGGC TCAGATTCAA GATTCAAGTT CTTCCACTAT ACAGCCAATT TGAAGTCAGC  1141 CCAGACTACA TGAGACCCTG TCTCAACTAA GCAAATGAAA GCAAACTGGG GTCCAAATAG  1201 GCACTATTCG ATGTTTTGAT GCAAGTTTGT GACTGAGGAG TGGAGGTGGC AAATGAAGAC  1261 TTTTTTCTTC CTCTTCTTCT TCCTCCTGGG TCCCGTTTTT TTTAGGGTGT TCTTAGGATA  1321 TGTATGTCTC ATTGGCACTA CTAAGAAGTG TGGGGTCTAG GGAACTTCCT GTTATGTATA  1381 CAAGGTAATC TTCAAACAAT TGTGTGGGCT GTTTTGGTAA CTACTCAAAT AATGCTATAG  1441 AAAATTGTAC AATATATTGG GGAAGGAAGG GAGTTTTACA CAGGAGTCAA CATGACTCTT  1501 GTCTCTGGAA AGCAACTTGT GATCCAATGA GGAGCTAAAT TTAGAGACAC AATTCAGGAA  1561 GAGAATCCAA TCAGAGCTTC CTTGTAAAAC AACTCACCTT CACAAACAAG TTCATTCCTA  1621 ATCGAATTTA AGGTCTAGAA ACTGCCAACC TATTAATGTT TCTATAAATA CACTTGGGGT  1681 CAACTACGTA GCCAAGGAAA TCTTTAATAA ATTGAACACA AATTGTCAGG GGAAGGTTAT  1741 TGCTGGGACT CCTGGAAGCA TGTATAAGCA GGGTAGGGGT GACATAGGGG TGGGGGGCAG  1801 TTAACTCACA GATATTAGTC TCAGATATTA ATGGCTTGTG TGTGAGCTGT CTGCCACACT  1861 TAATGTCAGT CACCTTGCCC GGAACTATTT TTCTCTCTGA TTCCAAATGT AGCTATTGGT  1921 CTATTAAATG ATTAACTTCC ACAGAAACTG ATAATATCCT TATGGAATCT GACTGTGGTA  1981 AGCCTGTACA CCCCCGCCCC AATTTCCTTC TAGATTTAGA ATTCCATTCC ATGAGCCATC  2041 ACACCCACGC TGAAAAAAGA AAACCTGTTG AATCAAATTT GTGTTTTGGA GGGTAAGAGC  2101 CACCCTTCCA ATTTATAAGG CTGTCTATTT CTTTGGGGGG GGGGAAATGA ACCAGTATCT  2161 TCTATTAGTA AAAGGAGTGT TTGAGCATGG GCACTACAAC CCACTTCTTT CAGGGAGATT  2221 CATTTTTCTC TGAGAACTCA GCCTCTCTGT GCTGGTGCCA CAGGAATTCT TAAACTCTTT  2281 CAACTCTCCA ATTAACCAGA GAGCAAACCC AGCACTTTCC ATCTATGAGA AATCTACACC  2341 ACTCATGGAA TCATTGTGTG CCCTCTCTCA CTGCCTAACA GGGGTACCCT TGCCAAAGAA  2401 AAGCAACTTA ATGCCAAAAA GGTGCATCAC CTGGCACTGC TTCCGAGGAT GGGCAATGTG  2461 CAAGCACTTT GTTCAGTGGC TCTGCCTTGG GGTCTCTTGA GGGGCGGCAG GTTACCTGGG  2521 GTGGGGGCGC ACACTCTCTG AAGGTGGGCT GCGTTCAGTT TCCTGCTTCA GGGGCTCCTT  2581 CATAGTACCG CCCCCTGATG AGTTTCTGCT CAGACTGGAA GGTGTCAGGT CCCAAAGAAA  2641 CCTGGGACAA GGCTCACTCA GTACCTGTCG CTTCTCCCAG CACGTCTCAC CCCACCCCTA  2701 CCCTAAACTT CTCTAGCCCA GAGGCTGGGC TCCCCCTTTC TCTTTCCTAC ATAACCCTGC  2761 CATTTTAGCT GTGAGCTCTC TCCGTCTTTA GCTCCTCTAC TGTTCTTTTA TCCTCTCTTT  2821 TCTCTCTCCT CTTCTTCTCT CACCCCCACC CCCACCCCCA TCTCTCCCCC CATGGTCTGG  2881 TTCAGTCTGG ACCCTTTCAG ATGCCTCTGT CTGAACTCTC CCTCATATCT CAATAAAACC  2941 CTTCTCTTCA GCCACGCCTT GGAGAGGTCA TAGGCTCATT TTCGTTCAGA AGGCCTATCA  3001 AAGAATCTGT GGGCTTATCT TTACATTCAC AATAGGCAGC TTGGCCCTGA GACCACAGTC  3061 CAGGTTAAAG TGTTACCTTG GAAAGAAAGT CTTTTATTCA AGGTGTCTGG TTTCTTTTCT  3121 TGTTTTTGTT TTTGTTTTTG GAGACAGGGT TTCTCTGTAT TATTTTGGAG GCTGTCCTGG  3181 AACTCGCTCT GTAGACCAGG CTGGCCTTGA ACTCACAGAG ATCCGCCTGC CTCTACCTCC  3241 TGAGTGCTGG GATTAAAGGC GTGAGCCACC AACGCCCGGC TCAAGTGTCT GGTTTCTTTT  3301 GATGTCTTTA GTTTCTTTAA TCCCATAATT CCTTTAATTA TACCCTCTTG TCTGTCGGAG  3361 AATGACATCA AGGATATCCA GTTCAAGGTT TCCTATGTAG TTCAGTCATA GAGTGCTTGC  3421 CCAGCTGCCA GACTCTGTCA GATGCCCAGC ACCACACACA TACAAAGCAT TTCCAGCTCT  3481 GTGTCTGTGT CAATTACTCC TGTCTGCTTC TCCATCCCCA GACACCAGGA GGGCCCACAA  3541 GAAGCTTGGA GCAGGGAAGA ATAAAGAGAC AATATCCATA GACACACAAA ACCTCCAAAG  3601 TACTTATGCA TTGAGGAATT ACAGCTTACA AATCCAGTCA CAGTATCTAT ATTCATGTTA  3661 GCCTGATTTC AATCCCCCAG CTACATATTC TTCCATGAGC TAGCTCCTTT CCTATTCAAG  3721 ACTCCCTTGA TAATAGTTGT TATCAGACTT TACCCCTATT AAAATATTTG GACCGTTTGA  3781 GAGCAATAGC TCACCTCTAT AATCTAGAAC CCAGGAAGTT AAAACAAGAT GTTTGCTGCA  3841 AGTTTGATGC CAGCCTGGGC TACATAGCAA TTTCCAGAAC ATCCTGAGCT ACAGGGCAAA  3901 ATTCTATCTT AAAAAACAAA AAGTAGACAG ATCAGGTGTT TCACCTTGTT TCAAAAAATG  3961 CAAAAAATAT TTTTTAATTG TAGAAATATA TACGCTAATT CCTTTGGTAC CCTAGGCCAA  4021 GTGACTAGAT GGGTTAGTCT TCCTTCTGGT CCTCACAGAA GAAAGTTAAG TTCTCAGCAG  4081 GAATAATAAA AAATATTAAA AAAAAAAACA AGCTGCAAAA TTCTGTTGTG GTTCTGCCAA  4141 AGTGTTCTCA GGAGTGAGGG CATACTGGGA TTTAGTCAAG CAGATATTTC TGTTTGAATA  4201 ACTAGGATCT GGGAGCCATG GGACACCACC CCCACCCATA AGGGCTACTG AAAACCACCC  4261 CTGGAAATCT GTAAATATTG CTAAGGCTCT ACCCTTTTGC TCAGAGAACA ACCACCCACA  4321 AGGATAGGGG ATAAGTTAGT TCTGTAGTAG AGTGCTTGCT TAGCACACAG AAAGTCTTTC  4381 TCTCTCTGTC TTTCTCTCTG TCTCTGTCTC TGTCTCTCTC TCTCTCTGTC TCTCACACAC  4441 ACACACACAC ACAAACAAAC ACATGAGTGC ACAAGAAACT TCTAGGTGCT ACTAAACTAA  4501 TGTAAAATCA TGCAAAGTTC ATAGAGAATT CAACAGCTAG TGACAGGATG ACCCGAACAC  4561 AAGATTCTGC CCTAGTCCTT GTATTCTGTA GTCCCCAGTT TCTCTTTACT GCCACAGTCT  4621 CCTATCTCTG ACAGCCTCCC TCTTTGCAGA TCTGGCAGTT TCTGGGCCTG GAACTGCTTT  4681 GGTAGAATGT CTGTACAGCA TGCACTAGGC ACTGGGTTTG ATCCCCAGCA CTGCATAAAT  4741 CAACTTTGAT GTCACACCTA TAATTTCAGC ACTTGGCAGG GATCGAAGCA GGAGGATCAG  4801 AGGTGAATCA AGGCCAGCCT GGGCTACTTG AAACCCTGGG GAGAGGGATA GAAGAAGGGG  4861 GAGGGGGGAG GGAGAAGAAA GGAAGGAGGG GGAGGGAAGA GGAGAGGAAG AGAGGAGGGA  4921 GAGGGAGGGA AACAGGGAGG GAGGAAGAGA AGGAGGGAGA GAGGGAGGAG GGAGGGAGAG  4981 ACTAGTGTAA GCAGAACCTG TAAGTTCTCT CCTCAGCCTC AACACACCCC AGCTCCCTGC  5041 TGTCTCCCGG TCCAGGGCTT CAGGGCCTGG CAGGACAGGC AGCAGGTTGT TTTGCTCTCA  5101 TAAAGCCATG TTACATAACT AACTAATGTT TTGAGCAGTG GAGCTGAGCC AATCTAGGTC  5161 ACATCAAGAG GGAATGGGGA AAGAGGATGA TCACGGAAGT GGTGAGAGGA AGGGAAACAA  5221 GAAGGGAGGA ATAAAAAAAA GAGGCGAGAG TGGAAATGGG GTGCGATTAT TTAATATCTG  5281 CTGCCTGTTC ATAGTTCCTG GTCCTTAGGG ACAGCATATA TTATCCTGAA AAGTCCTCTC  5341 TCTATTTTAT CTAGGCATTC TGTCATCCTA TAGCCCCCAC TCTGGATGGC TGAACTCTGT  5401 GCCAGCAGCC TGCAGGTATC ACCCCTTATT GGAGTGAGGT CTATTCCTTA TTGGAAGCAG  5461 TGGCAGGCTG GTAGGAAACA AACAGGCCTG GTGTTGTGGA ATGCTGTCCT CCCAGCATGA  5521 CCATCATTAG ACCTTATGGA AGCAGAGCGA GGGGGGCATT GTCCTCCTCC CCAGGCTCCT  5581 GCAAGCCTAC TCAGCTCAAC TGGTTCCCCG GGCCAGACTT AGGTGCAAGA GTTGCTTTGG  5641 TTTGTTATTG GTGGCCTGTG TAGCTGAGTA GACACATGCT CACCTACATG ATATATGATG  5701 GCTTGCAACC TTCTAAAAGT TCAGTTTCAG GAGATCCAGA ACCCTCTTTT GCCCTCCAAG  5761 GACACCAGAC ACCCATGTGG TACCCATACG TACATGCGGG CAAAACACTT GTGCATATAA  5821 AATAAAAAGA GATGGCTCCG TGGCTAAGAA TGCTCCCTAC CTCCAGCTCA CCCACATCTT  5881 CACAACTGAC TGTGAATCCA TCCATGGTTC TCTTCTGACC TCGGAGGGCA CCTGTGCCCA  5941 TGGGGCATAC ACATACACAT ACACAAAACA AGTATGTAAA TAAATAAATA TTTAAAATTG  6001 GGGCTGGAGA TGGCTTAGTG GTTGAGAGCA CTGGCTGATC CTCCAGAGGT CCAGAGTTCA  6061 ATTCCCAGCA CCTACATGGT GGCTCCCAAT CACCTAAAGT GGGACCTGAT GTCCTCTTCT  6121 GACATAAGGT CATACATGCA GATAGAGGAC TCAAATGCAT AAAATAAATA AATAAATCTT  6181 TAGAAAATAA GTACATAATA AATAAATATT TAAAATGACC CAAATTAAGA AAAAAATGAA  6241 GCCAGGCAGT GGTGGTACAC TCAGAAGGCA GAGGGAGGGA GATCTCTGAG TTTGAGACCA  6301 GCAGTTCCAG GACAGCCAGA GTTACACAGA GAAACTCTGT CTCAAAAAAA AAAAAGAAAA  6361 AAAAACAGAG AAAGAAGAGA GGAGAAAAAC AAGAACAAAA AATAACAAAA CAAAAACATG  6421 GCTTTCCCTT CATGGCATCT GCTTCATCTG CCTATTTGGT AATGATCAGG GCACTACACA  6481 CCCAGTGCTT CATACCCTGG CCATGTTTCT GTTCTTGGTG TCACCACCAA GTTTACTAAA  6541 GATGGTTCCA GAGTGACATT AGCAGCCCCA CACCCCAATT GCAGCTAGCA GTTGAGGAGA  6601 TTTCTGGCTT TTTGTCTAAG AGGAAGGTTC TTTGGCTAGG AGATATACTG AGAAGGACTA  6661 GGAAAAGGGG TGTCTAAGAA ACTTGGAGAG CACATTTTTC AAGTCAGAAA GAACATAGAC  6721 ATATTCTGGG GGTGGGGGTA GTAAGATAAT GGACCCTCCT AAGGGAAGGA TTGTGGGGTT  6781 TGCCTGAAGG GGCTGAAGCA GACCACTGAG GAGGCGAGAG CACCAGCAGC TTTTGAGAGG  6841 TGGGAACACT GCAGCTGAAG TCACTTGTCA CCTTCCCAGG TAGTTCTTAC TTCCAGCTCT  6901 GGCAGGGCTA GATAGCCTAG GAACTCCCAG ATAGGAGTTC TAGTTCTTCT TCTCCCAAGC  6961 TGACAGAACG TGAGCTCAGA GTCTAGGGAC ACTCCAGGTT AAGGACGGGG CCATTCTTGA  7021 TTGTCAGCAC AGATAGATTT TAATTAGAGA GCAATGACAT GACAGATAAA CAGCCCCTTA  7081 TCTAAAGGGG TACATCCCAA GACCCTGGAG GACTCTTGAA AACCCAGATA GGAGCCAGCC  7141 AGGGAAGCAT ATACCTTTAA TCCTAAGATT TGGGAGGCTG AGGTAGGAGG ATCTCTGTGA  7201 GTTTGAGGCC AGTCTTGTCT ACAAAGTGAA TTTTGGGACA GCTACACAGA GAAACCCTGT  7261 AAGAAAAAAA AAAAAAAGAA AGAAAGGAAG GAAGGAAGGA AGGAAGGAAG GAAGGAAGGG  7321 AAAGGAAGAA AAAGATAAAG GAAGAAAATC CAAATAGGAA AGAATCCCAT ATATACCATA  7381 TTTTTCTTAA ACATACATAG GTTTATTCAT TCTCTCTGTG TCTGTGTGTC TGTGTGTCTG  7441 TGTGTCTGTG TGTCTGTGTC TGTCTGTCTG TCTGTCTGTC TGTCTCTCTC TCTCTCTCTC  7501 TCTTTCTCTC CCTCTCTCTC TCTTTCTTGT CTCATAAATC TCAACACTCA GGGACCCAGA  7561 AGATATCCCA GTGGTTAAGA ATACACACTG CTCTTGCAGA CCTAAACTCA GTTCCTTGTC  7621 CCTACTTGGG GCAGCTCACA ACCACACCTG TAAGTCTAGC TCCAGGGAAT CCACACCTTC  7681 TGGCCTGTGC AGGCACCTGT GTGAAGGAGC ACATATCCTT CCCCATAATT AAAAAACAAT  7741 CATTGAAAAA TAAAACTCAA CCCCCTCCCC CGGGACTCAA ACCAGAGGTA GTCTCCCTGC  7801 CGTAGGCGCT CAAAAACTGG ACTTTCAGGT GTGAGCCTCT AGGCCAGGCT GCTTTTCTTA  7861 ACTGGCTACC GTGCTCTTGC CTGAAACTTC CAGCTTGAGA CCTCATAGTA AAAAGAACAT  7921 ACACGTCTTC TGTCTGTACT ATTTTACAGA CGGCTGACAT GTTCATACCA CGTATTTTAG  7981 CAATTTCAGC ACTTGGTATA TTTTCTGTCA TTCTCAAATA ACTTTCACCT TGCCACTTAG  8041 GGCAGTCCAA GGCTCCTCTT AGATATATCC AAATTATCAG CCACCACTTC TGCCTTTACT  8101 AAGTAAGACA GGGTACTTAA CATGGAGTAC TTAACACAAG CACTGTGATC TGAAGGTGGA  8161 GACTGCTTGC TACTCAGTCA CAGCTTAGCA TTGCTAGAAC AAATCCTGAA CAAAGGGTAA  8221 TTCATGACCC AGGCAGGGCA GAGGCGGATG GCTGTTCTTG CTCCTCAGAA ACCCCTGTGT  8281 ATAATTTCAA GCTTAGGAGT TGTTTGTCTT TGGATGGAGA GGGTCAGACC TAGGGCTTCA  8341 CTCACACTAG GCAAGCACCG CAGGTCTACC TTCGAAGAGA AGAATTTTCA CTTAGCGTTT  8401 TCAGATATAG GTCAACCTCA GCTGGCTGAA ACTTTGACTA AGTGAGCAAC TGTGAGGGTG  8461 GGGAACACAT GCATGCATTT CTTCATGTTA TAACATCTAT TTATACATAA ACATATCATA  8521 TAAATATATT CTATTGCATA TAAATATACA TAAATGCACA CTCATGTATA GATATCAATC  8581 ACATAATTTA TGCTTTTATT CATAGATTAT CTCTGGGAGG TGTACAATTA CTGACAATAC  8641 CTGCACATGA TAGTACACGT TGTTCTAGTT AGGTTTCTTT TGCTGTGACA AACACCACAA  8701 CCAAAAGCAA CTTGCAGAGG GAAGGGTTTA TTTCAGCTTA CAGTTGTATT CATTATGAAG  8761 AGTTGGGAAG TCAGGACAGG AACCTGGAGG CAGGAACTGA AGCAGAAACC ATGGAATAAT  8821 GCTGCTTACT GGTTTACCCA CCATGACTCA ACCTGCTTTC TTATATCACC AGGACTGCTT  8881 GCCCAGGGAT AGAACCACAC ATGGGGACTG TACCTCCCAC AACAATCATT GATCAAGAAA  8941 TGCCCTAGAG TCAGGGATGG TGGCAAATGC TTTTAATCCC AGCACTCGGG AGGCAGAACC  9001 AGGCCTTGAC TGTGAGGTCA AGGCCAGGCT GGTCTACAGA TTGAGTTCCA GGACAGCCAG  9061 GGCTACTCAG AGAAACCATG TCTCATGGAA AAGAAAAGGA GGAGGAGGAG AAAGGAGAAG  9121 GAAAAAGAGG AGGAGGAGGA GGAGGAGGAG GAGGAGGAGG AGGAGGAAAG AAGAAGAAGA  9181 AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA AGAAGTAGAA GAAGAAGTGT CCACTGGACA  9241 ATCTGATGGT GGCGTTTCCC AATTGAAGTT CCCCTTCCAA GATAACTCCA GGATGTGTCA  9301 AGCAGACAAA AACAAGAACC AAGACACATG TTTATAATCC CAACACTGGG GAAGTGGAAT  9361 AAGAGGTTTG GCAGTTTAAG GCCATTTTCA GCTACATAGG GAGTTCCAGA CTATCCTGGC  9421 TACATGAGAC CCTGTCTCAA AACACCAAAA TGCAAGGGAA AAACAAAAAG CAAAATAATG  9481 AGTACAAATA GCAGTGACAT TCTGGGGAGA CAGCCTGGAG GGGGGGATTG CTTATTATCT  9541 CTCCCTACCG TTTGGAGTTT TTAAAATCAT GAATCTAACC CCAGAAAAAA AAGCATTGAG  9601 ATTCTGGGAC ACTCGGGTGG TAGAGAAGAT CATCTGATCC TGTCACCTTT CGGGTACGTC  9661 ACTTTATTAA TCTCTCTGAG ATTCAGTTTC ATCACCTCTG AAGTGGTTTG TGTCGACGTA  9721 CAGTCCTCAG GACTAAGTAA GGCCACTTGG TGGCTGTGCC AAAGCACTGT GTCAGGGACA  9781 CGGCAGATGT CTGACACATC TTGTTAGATT CCTTTTCTGT CCTCCGCTCC CCTACCCCAG  9841 AGGTGGGTAC AGCCCCATGG CACCTCATCT TTAATGGCTT GGGTTTCTTT TCTCCAGCCA  9901 GGAAAGTTGT CGCTTTGGTG ACAGCTATTT TAAGTCAACT GACCTTTCCT GCAAATGATC  9961 CAGATGCCTC TATCTTAGGC TGGTGATGAC GAAGATGGCC TATGACGGGG TTCCTGGGGG 10021 TGTGTTGGGA GGTGGGGCAG GGGTGGGGCC CGGCATTTGT CAGACCCATA TGATCTTCTG 10081 GCTCCCGGGC TCTGCAGATT TCTCCTGCTG GAGATGCCTA CCTGCCAGCA ATCTTGGAGA 10141 AGACAGAAAT AGCAGCTTTG GGTTCCAGGT CCCCTCCTCC CTTTGGCCCA ATGTAGCTAG 10201 AGCTTTGGTT TCCTGCTGCT GTCTTGGTGC CTGGAGCCCT CTCTGGATGG TCATGGAGTC 10261 TTGTCAGAGA AGCAACTTTG GGCTGGCAGA CAGTCATTCC AGAAGACATG ATCTGGAAAA 10321 ACTGCTTCAT CGTTTCCTTC AGAGGCACTG TCCCGAGCCC ATTTCCTTGT CTGGTTCCTG 10381 AAATCTCAGG GATGCCATCA GAAGAAGGTG TTCTTGTGTT TACTTTGGAC ATGGTTTTCT 10441 GTAGTGCAGA CTGCCCTTAA ACTCTACGTA GCTGAAAATG ACCTTGGTCT CCAGACCTCT 10501 TGATCTGTCA GCATCCCTGG GAAATCCAGG GTTCTGTAAT CCTCCCCTCT CACCTTGACT 10561 TACTGTACCA GCATCAAACA TCCTAAACAA ATCCAGTGTT TAGCCAAATA CAGCGGTGCA 10621 TGTCTGTAAT CCCAGCCACC TGGGAAGCCG AGGCAGAAGG ATTAAGGGAG CTGGAGGCCA 10681 GTCTGTGCAA TTTAGCAGGA CTGTCTCAAA ACAAAATTTA ATGGTTAGGG GTGGGCATGT 10741 CATTTATTTG ACTCTTATCA CATGAACACA CCTGTAATCT CATCACGAAA CGACAAGGCA 10801 GGAAAATCAA AAGTTCAAAG TCATCTTTGG CTACATAGCA AGTTCTAACC TGACCTAGGG 10861 TATGTAAGAC CTTGTCTCAA AAGCAAACAA ACAAACCCCA AATAACAACA ACAACAAAAC 10921 AAAAAGCAAA CAAGGAGAGG GTGTGCAGCT AGGGATATAA TTCAATGGGT GAGGGCTTAC 10981 CTCACATGCA CGAGGCCTTG GTTTCAACTT CCAGTTGAAA TGAAGTTTAG TGGTAGAGTT 11041 CTGTGCAAGG CTGTAGTTTC AGCTCTCCAT ACTGCAAACT GGAAAGAACA ACAGTGACAA 11101 ACAGAAACAA AAAACCCCCA CAAACAATGT GCTTTCTCAC TCAATAAAAC CACCTCTTTA 11161 CATACAACTA CAACTGCTAA GAAAGTTCTT CAGTGTTCTA GAGCCTGAGC ACCTCAAATG 11221 GTTTCCATAA AGCTGTATGC AAACACTGAT AAGCCACGAG AAGCAACTGT ACAAAGCACC 11281 CTTTGATTTT CATAGTTTAT CTACACAAGG ATTCTAGGAA AGTGTGCTAG GAAAATTTTA 11341 TGTATCAGCC TTGCGGGTTT GTCCAATAGT TTTAGATTTT GCCAGTGAAG ATTTTCCTTT 11401 CTTTATTTTT TACATGGGAA GGAAGTTTAA TTGGGGGAAG GGACGGGAGT GGGCTTTATT 11461 TTTATTTTTT AATGAGACTA GCATTTGCAT TGGTGGACAT TGAAGGAAAC AGTTTCCCCT 11521 CCCTAATGTG TGTGGGCCTC ACCTAACTCA TTGAAAGTCT TAGATAAAAC TAAGCTGAGT 11581 GAGTGAGTTG GCCCATACCT GTAGATGGAA GGAAAAGGGT CTTGAGTTTT GGTTTATCCT 11641 AGAGAGAACT TGATCCCCCA AACACCAAAC TTTCAAACCA AACCCCAGCC TCCTCAGTGT 11701 GAAGGGATGC TGTTACATGA CCACCTATGG ACTCAGACAA CCTCTCTTCC CTGAGTCTGC 11761 TGGCTTACTC ATCAGAGTCT GGGCTCACGA AGCCGCCACA CATATATGAG CCTCGTTCTC 11821 CCCACTCTTC TCTTGTGGCA CTGAGGTTCA AACCAAGGAC CTCGCACATG ATAGCAAATA 11881 CTGTACTGAA CCATAGAGCC AGCCCTTGTC AGTTTCTTAA CACAAACATA TAGATGTATA 11941 TGTATATGAA TATTTCCATG CTACCAATTC CATTTTCTCA GAGAACCAAA GAATACACCA 12001 AGTAGTCACA CTTGAAATTC TGTTCTGAGA TTGAATAAAA CCTGATCAAA TGTGAATTCG 12061 GTCCCTTCTC CCCCATCCCT GACGCCACCA CGTTGCTATA CAGACCAGGC ACAAACTCTT 12121 CTCCTTGTGA ATGTGTGTAA CACATGTTAC CACTGTGCTT GGCTTTTGTA GTTAGAAGGT 12181 TGGTTGATAT TTAAAAAAAA ACTTTAATAT TTAGTCATTA CTTTTTAGTA AAGATTTGCC 12241 TTGCTTTTAT TTTATTCATG TGCATGTGTG TGTATCTGTG TGAGTGTATG CCACGTGTGT 12301 TTGGGTGCCT CTGGAGATTG GAAAAGAATG TCAAAATCCC AGGACCTGGA GTTCCAGGCA 12361 GTTGTAAACT TCCCAATGTG GGTAATTATA ATGAACTTGG ATCCTCTAAA AGAGCAGAAC 12421 TCACTCTTAA CTGATGAGTT ATCCTTCTAC CCCCAAATTT ATTTGTTTTG TTTATTTGTT 12481 TATTTATTTG AGAGGGTCTC ACTGTGTAGC TCTGACAGTA TTAGAATTTA CTATGTAGAC 12541 CAGACTTGAT AAATGTCTAA CCCTAGAAAA AAATAGTTTT GTTTTGATTT TATGTCTGTG 12601 CCATCCACTC CTTGAACATA TATTTGGTAT CTGTGAAGCC AGTGAAGGCT GTTGGTTCCC 12661 TTAGGACTGG AGTTACAGAT GGCTCTGAGC TACCATGTGC ATGCTGGGAA ACAAACTCAG 12721 GTCCTTTGGA AGAGCAAAAA ATGTCCTTTG ATGGTGGTGG TTTGAATGAG AATTGCCCTA 12781 TCGAGCATAA AAACTTGGCA GCTTTGGCTA CATGGTTCTG GATTAAGAGT CAAGAAGGAT 12841 ACAAGAAAGC GGTTGTGGAA TGATCCCCCA TGGTTAAGGA AAACCACCAA AGCCAGGCTT 12901 GTGGCAGGGG AGTTCCTGCA TGGAGGCCAA GAGAAGCCAC TATGTCAAGC TGTGAAGGTG 12961 AAGCCTGGAT TGTGTTGGAG ACCCAAGCTA CTGGAGATGT AAGAGATGTG AGATAATGCC 13021 CAGGAGAGCT GCAGACAGGG CATGGAATCA GGCCAAGCGA GAGAAGTGTG TTGCAGTCAG 13081 CAGAACTGGG AGGGAAGAGT CATCTAAGTC CTTTGTCATC AGACATAGAG ATACAGGATC 13141 TGAAATTTGC TCTGCTGGGT TTTGGTCTTG ATTTGGCCCA GTACTTCCTA ACTATGTCCC 13201 CTTTTCTCCC TTTTAGAATA CTAATTTATA TTCTGTGCCA TTGCCGGTGG ATCAGGATGG 13261 TTCTCAGATA CTGTTTTAGT TCCATGCCTG TCTACTTCCC GTCATGACAG TCATGCACTA 13321 ACACTCTAAA ACTGTAAGCA AGCTCCCAAT GAAATGTTTT CATTTATAGA GGTGCCTTGA 13381 TCATGCTGTC TCTTCACAGC AATACAACAG TGATTAAGTC AGCTGCTGAG CAATCTCTCT 13441 GGCCCCAGAA GTATGCATGT GTGCAATTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT 13501 GTGTGTGNNN NNNNNNNNNN NNNNNNNNNN NAGGAAATGT CATTCTGTAA ATATGTTTAT 13561 CTTATTGGTT GATGAATAAA ACACTGTTGG CCAATAGGGC AACAAAATAG GTGGGGCCAG 13621 GATATAAGGA GGATTTTGGG AAGTGTAGGC AGAGGGGAAT TGTCATATGA TCCCAGGAAG 13681 AGACATAGAT GGGCAGAAAC TGCCTCTAGC TAACCATAGA GGTCTGGAGG TCTGTACAGA 13741 CAGGCAGGAA GTGATGTAGC TGGAAGAATC AGAATATAAG CAGGAACAAA CAGGAAATCG 13801 AGCTCTTCTT CTCTCTCCAC TTCAGAGATG CTGAACAGTT GAGATGCAGG ATGCCAGAAG 13861 AGTAAGAGGT CCCTGGACCT TTCTCCAGTA AGATAAGACC ATGTGGAAAT AGATTGATAG 13921 AAATGGGTTA GAGATTAAGT CAGAGCTAGC CAATAAGAAG CCGTAGATAT TGGCCAACCG 13981 TTTCATAATT AATATAGCAT CTGTGTATTT ATTTGGGGGA CCTGGTAGAC CAGAAAACTC 14041 GTGTTAGAGA CATCTTATCA AAGTTGAAAA AAGAAAAAAT GTGATAAAGT TAGGAAAAAA 14101 TATAGTAAAT GTTAAAAGCT AAATTCTAAA ACTACAACTT ATTTATCATT TCCTAAATGT 14161 TTAAAAATAT TATTTTATAA TGAAGATACT TAAAATTCAT TTCTCTGTCT TTTGAGACAG 14221 GGTCTCAGTG TCCTGGAACT CATTATATAC AGCAGGCTGG CTTGGAACTC ACAGAGATCC 14281 ACCTGCCTCT GTCTCCTAAA TGCTGGGATT AAAGGTGTGT GCCACCAAGC CTCAATTAAA 14341 ATGCGTTTCT TTTTCTTTCT TTCTTCCTGT CTTTGATTTT TTTGTTTGTT TAGATTTTTT 14401 TTTTTAGACA GGGTTTCTCT GTTAGCATTA GTTGTACTGG AACTCACTCT GTAGACCAGG 14461 CTGGCCATGA ACTGAGAGAT CTGCCTGCCT CTGCCTTCTG AGTGCTAGGA TTAAAGGCAT 14521 GCACCACCAC TGCCAGGCTT AAAATGTATT TCTTTTTTTA ATTTAGAAAT TTATTCTGTT 14581 TAATCCACAC GCTTTATATA GCTTTAGTTA AGAAATAAAA TAAAATGAAA CAGTGAAACC 14641 AAGAGACTAT GTCCAAGTCC AGGTCCTCCC AGCCTGCCAA TGCCAAGAGC TCTTTAGTTC 14701 TGTGTACCAA TTGGAAGAGT AAGAAAAAAA TATGGATGGG AACCACACAG TTTCATAAAA 14761 CAGATTTATG GAACTGAAGG GTCCTTGCTG AGTCTAGCAA ATTGCCTTTA CAAAAGAGAA 14821 AGAAAAAAGG GGGAGGTAGA AAAACAAAAC AAATCAACCC AAAGAGGACA AAATCCCAGA 14881 GTTCTAAATT GACTTAGGAA CCTGTCACAC TGGGACAGAA GCTTCAGCAT CCATGAGCTG 14941 TGCCTCCCCT GCTCTCTAGA GCTGGGATCT CGAGGTGTCA GCAGAGACCC CACAGGTAAC 15001 AGGAGCAAAA ACACTCACTC AGACCTTTGT GGTACTTCAA CAGTGGTCTC ACTTCTGGGC 15061 AAGCTTACAA ACCTATACAA AGTTGAAGGT GTACTTTACA TGAGTGCTAA ACTTCAAGAG 15121 GAAGGAAGAA AAAAAGGGAG GTGGAGGGGA CAGAGAGAGA GAGAAAAAAA CAAAACAAAA 15181 CAAAAACAAC CACCTCAGGA GAGGCAAGGG CATTTAAAGG AACCACAAGA ATGCCAACGA 15241 TATTAAAATG TATTTCTTAA TAGTAAATTT TATGGGAAAA GAGAGTCTCC TCTTCCTCCA 15301 AGTAGGCTAG GTAAGTACCT TGCCACTGAG CTCTATCTAT ACCCTTCAAA GTGGACAAAA 15361 TGACAAAGAT AGTTCATCTC CCCCAAAGGC CCTGTTGGGG TGCTGATTGT CACATCTGGT 15421 GAGATTTCTG TTTTTGTTTT TATTTCAAGA CAGGGCCTCT CTACATAGAT AGTCCTGGCT 15481 GCCCTGGAAC TCACTCTGTA GACCAGGCTG GCCTGGAACT CATAGACCCA CTTGCTTCTG 15541 TCTCCCAAGT GCTGGTGCTA AAGGTGTGCA CTGCCACTCT TTTTAAGTAA CTATGAGTTT 15601 CAAAACAAAT TAAAGAGCAC TGTTAAAGTG GCTTGTTGTG TAAGCCTAGC TTCAAGTCAA 15661 AGGCCCGAGG CTCCCCTACC AACCAGCTGC TATCACCTAG ACACTGTCTG TAGATCTTGC 15721 ACTGACTCAA AACTGTGGCC TAAGGTCAAA ATAATGGTCT TCCTGGATTC TGATGTGAGT 15781 GAGATTGTGT AGGAGGGCTG GCCGCTGGCC TGGCTTGAGT CACTCTCAGC TGGTTTCATC 15841 CCATTCCTGC AACTCTGTGT AAGAGGTGGA TGATCCTTGC TTAACTGATG AAGAAACCAA 15901 AGCTGTAGAA AGGATCATTT GCTTAACTCT TCACAGATGG CAAGAGGCAG AGTCAGGATT 15961 GGCAGAGTCA CTTCTGCCAA CTTCACCCTC CTGCTAACTC CACCCTCCTG CTAACTCCAC 16021 CCTCTTGCTT ATACTTGACA GTGGAGGAAA AGCCACTGAG GGAATTAAAA GTTGTTACTG 16081 GTAATGGTCA GGAAAAAAGC TGAACAAAGG AGATTAGATT CAGGGATCTT TTTCTGAAAA 16141 GAAAGAAAGA AAGGGGGACT ATAGTCTAGA AATGCTGAGA TAAAAGGGTG GATTATCATA 16201 TCTACTCTCA AACTAAAGAA GCAACTACTA GTCTCAAATA CTTTATATTG GTATGGATTT 16261 TTGTGTATTG GTACAAATTT AAGGTTATTT TTGTTATACT GTATATATGT TTTTCTTTCT 16321 TGTTTAAGGT ATTGTACCTG TATAGCTTAT TTAAAAATGC AATGTAAACA TATAGTCCTT 16381 GAAAACTATT TAAGATAATA AAGAAATACA GGTTAATAGT CATCTATAGC AATCAAACTT 16441 ATAGTCATGT TAGGTATGTT TTCAAGGGCA TACAGAAATA AATTTGAGAT AGATAGGTCA 16501 TCTTCAAACA CTCCAGAGAT CTACAGAAAA TGGCATTTAT AAAATGTTTT AATGACATAA 16561 GATTTTTCAT GATAGTGAGA AATGTCTACT CTTGGCAGCA CCAATTTACT TCAAAAATGG 16621 ACAATGGGCA TTGAAGAAAC TCCATGTGGA TTTTGCTTTC TTTGTGGCAA AAATCTAGCT 16681 ATCTGGGCAA GAAACTTCCC TTACCTTGAC TGCTGTCCTA ACTGGACAAG CAGGACATAA 16741 AAGAAATTGA CTGCTGAACT TTGCCAAGAT AGTATACATT AGTCTTTCAA AAATCCCTGC 16801 TTTACAAAAA AGTCTATCAG ATATTCTAAG CTTCTAGGCC AAAGATGGAT GCTTCAATGT 16861 TAACAGAGGA ATCTTCTGTG ACTGATGTTT CTGTCATTTC TATAGTTTTG AAAATTGCTT 16921 GCTCTGTTCT TCCCTGTTTG CTCAGGTAGT ATTATTTCCT TCTTGAGTGT CTAATGGAGT 16981 TAAAGACTAG ATAGTTATAG CTACAGTTTT CCTTGTAACC AAATTCAGAA AAGAAACTCC 17041 CAAAAGAGGT GTAAAAGTAT GAGGCTGAGA AATATAAAAA CTTAAATTTA TCTAAGAAAA 17101 TGTTTTGTTA TCTAAAAAAA AATAATTTTG GGTTAGTAAT ACAAGTTAGG ATAGAAAATG 17161 AATTAGGTAC AAAACTTTGG ACTCATCAAG AAAAAATAGA TAATGGAGTA TTTTCTCTGA 17221 ATTTGCCAAA TACAAATAGA CTGGGTATTG TAAATGTAAT TCTTACTTGA TAATTGTTCT 17281 TATTGTTTAT AGTTTATTAT GTTAGAGTCA AAACCTTTCT TTTTTATTTA GACAAAAAGG 17341 GGGAATGTAG AATATTTCTT TACACTGTGT GAAGATGTAT CACTGTGATT GGTTTAATAA 17401 AGAGCTGAAT AGGCAATAGT TAGGCAGGAA GAGGTTAGGT GAGACTTCTG GGAACAGAAG 17461 TCTCAGGGAA GGAAACAGGC TAGGTCACCA GCTAAATGAA GAGGAAATAG GACACTCAGG 17521 AGGAGAGGTA ACAGCCACAA GCCAAGTGGT GGAATATAGA TGAATGGAAA TGGGTTAATT 17581 TAAGTCATAG GAGCTAGTTA GAAACAAGCC TGAGCTAAAG CTGAGCTGTC ATAACTAAAA 17641 GTGGAGCTTT CATAATTAGT AAGTCTCTGT GTCATGATTT GGGGGCTGAC GGCCCAAAAA 17701 AGCCTGCTAC CCAAGTTCTT TTCAATTTTC AAGTTCTAGG ATTCTGGCCT TTTATTGGAA 17761 AACACTGTCA AGTTTCTATA GAGGTCTGAC TCCACAGTGT TGCCTGTGCA ATGAAATTTA 17821 TTTAATTTAT TCCGAGGCCT TGTGCACTCT GGATAATCAC TGTACCACTT AATCTATATT 17881 CCCATCCTTC ATTATAATTT AAAATGGTCT TATTAATCTG GTCACTTGGC TTTTTTTTTT 17941 TTTTTTTTCT GAGACAGGAT TTCTCTGTGT AGCCTTGGCC ATCCTAGAAC TTGCTCTGTA 18001 GACCAGCCTG GCCTGGAACT CACAGAGATC CACCTGCCTC CCCTCCAGAG TTCTGGGATT 18061 AAAGGCGTGT GCCACCACCT CCCAGTGAGT TTATGTCTTT GCAAATTATA CATGGTTTCA 18121 GTTTTTTTTT CTGTTTGTAA GTCACTTTAT TTCAAATGTA AAGTTTAAAA CAAGAAGCAA 18181 ATTACTATGA ATTTTTGTTA ACAGTCATTT TCCTTAACTA ATAAGTTTTA AATTTTCATT 18241 AATATGTTTT GATCATATTT TTTCCATGCC CCAACACCTC CAAAATCTCC CCACTCATTC 18301 AGTTCTTTCT CTATCTCAAA AAATGAAAAA TCCAAGCAAA CAACCATTAG ACAAAAAATA 18361 ACAAAACAAA ACAAAGCAAA GCAAAATAAA AGCACACGGG CTGGAGAGAT GGCTCAGAGG 18421 TTAAGAGCAC CGACTGCTCT TCCAGAGGTC CTGAGTTCAA TTCCCAGCAA CCACATGGTG 18481 GCTCACAACC ATCTGTAATG AGATCTGGTG CCCTCTTCTG GTGTACAGAT ATACATGGAA 18541 GCAGAATGTT GTATACATAA TAAATAAATA AAATCTAAAA AAAAAAAAGA AAAAAGCACA 18601 CAAAAAACCC AGAGAGTGTG TATTGAGTTG GTTAACCCCT ACTCCTCTGG AGTGTGATTG 18661 ATACAGCCAG TGCCGCTATT GGAGAACACT GATTGTCCCT GTCCTTACAG GTATCAATTG 18721 TGTGTAGCTC CTTGGTTAGG AATGGGGCTT TGTGTGCACT TCCCCTTTCA GCTTTGTAAA 18781 GGGTGTCCGA TTGAAGTTCG TATCTTCTGG GAGAGCATAA AATCAAAAAA AGATAAATGG 18841 ACTCCAGTGA AAAAGGAGCA AGCGGCACCT ATCTTTAAGG TAGAGAGGCA GAGGAGTGTG 18901 GTGTGGCCTG TCACAAACAC CCAATTCCCA ATCAGCTGGC GTCTACCAGG CTGCTTTCAC 18961 TTAGATGAAC CCTGACCTCC ATGTCTCCTT AACATTGCCA TTGTTTAACT GTTAGTGAGT 19021 CTGCCCTCTG TTCACTGAAA GACTTTCAGA AGGTGGTGTC GCCTGCCTTT AATCCTAGCA 19081 CTCGGGAGTC AGAAGCAGGT AGATAGAGCT CTGTGAGTTT GAGGCCAGGC TGGTCTGCAG 19141 AGTTCCAGGA CAGGCTACAG AGTGAAACCC AGTCTCACAA ACACCGCCTC CACCACAAAA 19201 AAAAAAGGAA ACAAGATAGA GTGAACAAAC CCAGCTACCT AGACATCTAT CTGGTAAACT 19261 GACTCATCCC AATCCTCCCT GCCCTCCCAA AGAGCTTGGC TGGCTCACTT CCCCAAATGC 19321 TCTTCCCCTT TAACATTTAA CTAGTTCTTG TCTCTTGTAT GGTTTCCTTT TAACTGTATC 19381 CACCACCCCT ACCTTGACTT TTGTCCTGGT TGGTTTTTAA TTGTAAACTT GACACACAAA 19441 GTCACCTGGG AAAAGGGAAC CTTAATTGAA GAATTGTCTT AGATTGGCCT GTGGGTGTAT 19501 TTATAGGGCA TTGTCTTGAT TGCCAATTGA TTCGGGGTGG GGAGTGGGAG GGTAGGGTGG 19561 GGGTGGGAGC AGCCCACTAT GGGACTCACT TTCCCTAGGC AGATGGCTAT ATTAGAAAGG 19621 TAGCTGAGCC TAAGCCAGCG GGTGAGCCGA GCCAGCAAGT AGCATTCTTC TATGGTTTCT 19681 TTCTTTCTTT TTCTTTTTCT TTTTCTTTTT CTTTTTCTTT TTCTTTTTCT CTTTCTTTTC 19741 TTTTCTTTTT TTTTTTTTCT TCCCGAGACA GGGTTTCTTT GTGTAGCTTT GGAGCCTATC 19801 CTGGCACTCG CTCTGGAGAC CAGGCTGGCC TCAAACTCAC AGAGATCCTC CTGCCTCTGC 19861 CTCCCGAGTG CTGGGATTAA AGGCATGCGT CACCAACGCC CAGCTCTTCT GTGGTTTCTG 19921 CTTCAGATTT CTGCTTTGAG TTCCTGTCTG ACTTCCCTCA ATAATTGTTT GTAACCTAGG 19981 AGTGTAAGAC AAATGAACCC TTTCATCCCC AAGTAGCTAT GGATTTAGAG TGGTTTATCA 20041 CAGCCACAGA GTGAAACCAG AACAACTTTC TAGTAGCCTC TTGTTCTACT CCAGCTGCTC 20101 CTCTGACTAT TCCTAAAAGG TAGTTGGGCT CAGGGAACCA CATCCCGAGA GATTCAGCCC 20161 ATATGAAAAT AGCTCCATTG TGTTGAAGAA ATGTGACCCT CCAGGATTTC AGGCATCAGG 20221 ATTCCATGTT GAAAATGAAA ACAATTATTT TCCTCTCTCT CAAGATTCCT TTAGTCACCT 20281 TCCCTTACCC CAGTTCCTGG CTTTCCTTCT AAACAAATGT TCAGGGAGGT TCAAACAAAC 20341 AGCTGTGAAG AGCAGCATCC CATACCCCCA CCTTCCGACC CAACACTTGC CAGTGCTATA 20401 AGTAGACTGG GATCATCCCT GGACACTGTG TTAAATTACC CATGACCAAC CTTCTAGCAA 20461 GCTCTCCTTT TCAGGATTTT GTTGTTTGTT TGGGTTTGTT TGTTTGTGAC TTGATCTCAT 20521 GTAAGCTGAC CTGGAATTTG CTTAATAGCC AAGGATAGAC TTACAACCTG TGATGCTCCA 20581 GCCTCTGACT CCTGAGTACC AGGGATTACA CATGTGTGGC ATCACAATGA AAGATTTTAG 20641 TTTGCTGAGA GAAAAAGTTT TTAAAGATTT TAGTTCACAG AGAGAATAAG TTTCCCACAG 20701 GCCTTGGTCC AGGACAAGGA AGTTGGTCCC AACCCGAGGG CAGACAAACA ATCCTTTTTG 20761 GGTCACACCT GGCTGGCCAA CAGACAATAA AGGACTTCTC AGGGTACATT CTATGGTTGA 20821 CCACTCTAAC ATGAGATCAT ACTTTGTAAT CAATCACTTT GTGCCCCTTG CCTGTATGCT 20881 GATCTGCGGT TTTTTACAGG CTCCTATATA AGGAGTCTGT AACCCTTGCT GGGGTGTGCA 20941 GCTTCCCCGA TATTGCTGAC ACCCGAATGA GCATTCGTTC AATAAACCCT CTTGCTTTTG 21001 CAGCTCTTGG TCTGGTTTCT GAGTCTTGGG GCCTCCTTGG GATCCTGAGA CCCTTAAGGG 21061 TCTGGGGGTC TTTCAACACT TAACTTTCCT GTTTTTAAGT AGGAAGATCT GAAATCCCAG 21121 ATTCCTGACT CCATTGCACA TTTTCTGTAT TAGAGGCTGT AGCTCTGTAT AGTGGGTTGT 21181 GTGGCTTACA CATGCTCTGA GCTGGAGATT CTAGGGACAC TTAGGGTAAA GTGGAGTGTC 21241 AGCCCCTTTC CCTGCTAGAC TGAGGCCTTT CTGTTCTTTC CTAACTGGGA GGCTGTATAG 21301 CACCCAATGT GTTCATTAAA CTCCATATGT TAGCACTGCA TGGAATCTGA CACACACACA 21361 CACACACACA CACCCTCTAC CACCACCATC ATCAGCACCA CCCCCATCAG CACCACCCTC 21421 ATCCCCCCAC CCCCCACCCT GCCCCNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNC 21481 AACTGGAGGG TAGCATTAGC ACCCAGATGC CATTAATGTG CCAAATATTT GCTTGCTTGC 21541 TTGCTTGTTT GTTCCAGCAT CCTTAGTGAA TGCTCCTGCC CTCCTGGTTA AAGATGGCTT 21601 TGGCATCTCT TGGCATCTTT CTTGTATTCT AGGCCTGAAA TAGGGATGAA TGGTGAAGGG 21661 CAAGGAGGTC AAGTGTCACT TACCACCTGC ACTTGTCCCT TTAAGGGGTT TCCCTAGAAG 21721 CAGTGTACAT TTCATTAGCC AGAGCTTTGT CACCTGGCTA CTTGTGAAGG AGGTGGTGAA 21781 GAAGCCTTAC CTTTGACTCT GCCACTTGGA GCCAAGTCAG GATTCTCTCC CTGGAAAGGA 21841 AATGGAAGAT TAATACCTTG TTGGTTGTTA GACCTAGCCC ATTATGCGCC ATGAGGAAAG 21901 AGAGACAACA GTGGGTCACT GATTGATCAG GGTTACAGGA CAAGGAGCCT TGTTTCTCCT 21961 AACAGCTCTG AGCGGAGACA GAAGTGGAGT ATATAGGCAT AAAATTCACA AACATTTGCT 22021 GCCACGTTAC AGGTACATTT TTTCACCAGT CAGAAATCAA AGATTAGGGA CTTTGCTTGT 22081 GTGTTCCATC ACTGTCAACT GACATACACG GCAAGCCTTT TAGTCCAACC AATCAGAATC 22141 ATTTGTTCCT TCTGTTGTTA GGAGCAGCCA TAATGATTCT AAAGAACTAA CAATGCATAA 22201 TGACTATTTT TGTAGTTTAG GGATGAGGTA TGTCAGCCAT TGGACAGTTC TCAGCTCCCC 22261 TAGGGCTTGG GAACTTGAAC TTTATTTCAT CCTGCATGTA ATGGAGTCTG AAGTCAAAAT 22321 GGCAGTACTT AGGTCAAGGT GCTCGTGCCT GCTGCCTTCA AGGTGGTTTC CCATTCCCAC 22381 CATACCAGAG ACTTCCTACT GCATCTCCAG TCAAGGACAC AAACACTTTT AAGTCCTGAC 22441 TGTTGATTCA ATCTATATAG TTACCAGCAT AGAGGCTAAG AGTCACACTG GCTTGCAGGG 22501 GACTTCTCTA GCATATGTGA AGCCCCGTTT GAATCCTAAA CACAAGAGTC TAAGCTTTGG 22561 AGTGAGAGAC AAGCATGTTC AAATCTGTAC GTCACCACCC TATAGACATA GACAAGTCCC 22621 TTGGGCTCAG TTTTTTCACT ACAGAGAGTA ATTGTTATTT CAGATTCCTA GGGTTGTGGT 22681 AATTAAATAG TTGAAAGATA TAGCCCATGG AACATAAAAA AAACTCAAAA CCAGGCACAG 22741 TGGCACATGT CTTTAATTTC AGCACTCAAG AGACAGAGGC AAGTGGATCT CTGTGAGTTT 22801 GAGGCCAGGC TGGTCTATAT AGAGAGTTCC AGGTCTACAC AGAGAAACAG GCTCAAAACC 22861 AAAGCAAAAG CAAAACCTCA ACTAATGTTC ATAAAATTAT GAAATTGCTG GTACCAGTGA 22921 CATGACTCAT TGGTAAAGAC ACTTGCTAGC AAGTTTAATG ATCTGAGTTT TATCTCCGGG 22981 ATCTACAATG TAGAAGAAGA AAAACAACTC TCAAGAGTTG TCCTCTGATT TCCACTTATG 23041 CAAAATAGGA TGGGAACACA CTTAAGCAGG TAGGTAGGTA GGTAGATAGA TAGATAGATA 23101 GATAGATAGA TAGATAGATA ATAGACATAA TTAAGAACGT TCAGTTGCAG CACAGTTCAT 23161 ACTGAACTGC ATTTGGACAC CTCTGTGAAA AGTCAGGAGC TCTCCTGTCC TCCTGGTGAC 23221 ATTTAAACAT TGAAGGCAAC TATTTTAACT GTCAGTTATA TACAAATCCA CTGGCCTTGT 23281 AAAATTTTAA AACATAACAG AGGAGGCTAA AGTCCTGTTT AACAACCCTC TCCTTTTACC 23341 ATCCCAGGAA GCCAAAATTG TTCACAATTT GTTCTCTTCC CTCAGGCCTT CCATATTTCA 23401 AATACCACAT AAAACACCTA TGGAAAAACA TGAGGTATTA AAAATGTCAC TTGGAAATCC 23461 TTCTTCAAAC AAGCTTGTTC TTTCTTTTTT CTTTTATGTA CAGTGAATGG AATCCAGGAC 23521 CTTTGCAGAT GCTAGGCGAG TCCTTTACCT CATTCCTCTT TCGATTTAAA ACTTTTTCTT 23581 GTTTTGTGGA GACAGGGTTT CTCTGTGTAG CCATAGATGT CCTAGAACTA GCTCTGTAGA 23641 CTAGGCTGGT CTCAAATTCA GAAGCCAGTC TGCCTCTGCC TCGGGAGCGC TAGGATTAAA 23701 GGTGTGGGCA GAGTGCTAGG ATGAAAGGTA TGCACACCAC CACTCCTGGT TGATTTTAAA 23761 AAGATGCTTT TTAAAAAAAA TGATGTGTAG GTAGTGGGGG GAGAGACGGT TTCATGCCTA 23821 AGAGCACTGA CAGCTCTTCT AGAGGACTCA GGTTCAATTC CCAGCACCCA CATGGCAGCT 23881 CATAACCATC TGTAACCCCG GTCCCAGGGA ATCCAACACC CTCTTCTGGT CTCTGTGAAT 23941 GACAGATATG CATGGGATAT ACAAACATAT ACGCAGACAA AACACTGTAT ACATTAAATA 24001 AGTACAAATT TAAAATATGT GTAGGCATGT ATGTCTGCAT GTGGGTATGT GTACACTGAA 24061 TGCAAGTTCA CTTGGAGGCC AGAGATATAT AGATCCCCTG GAGTTGCAGT TACAGATACT 24121 TGCGAGCTGC TGTGAGTGTG CTGGGAACCA AATCCTCTGG AACAGCAGCA AGTGCTCTCA 24181 CCTGCTGAGC CATTTCTTCA CCCGCTTCTT TCTACTTTTT ATTTTGAGAC AAGGTCTTAC 24241 TAAGTTATAT ATTCACTTGG GGCTTGAATT CATTTTGTCA GCAGGCAGAC CATAAACTTG 24301 CCTTCCTCTT GCCTCGGGCT CCTGAGTAGC TGAGACTTCA CCATGAGGTC TGGCTTTGAT 24361 TACATTTTTC TTTGTTTTCT TTTTGGGGGT GGGGCTGATC ATGAACTCTA AATAGCCAAG 24421 GATTGATAGT GAAGTCCAGA TTCCCCCACC TATCACCGGG TGGAATTACA GGTGTGCACT 24481 ACCACACCCA ATTTGGTTTG ATTTTTTTTT TTTTTTTCAG GACAAGCTCT CCTTTTATAG 24541 CTCTGACTGG GTTGGAATTT ACTATGTAGA CTAGGCTAGT GTCAAAATCA CAGAGATCTT 24601 CCTGTCCCTG CTTCCTGAGT ACTGGGATTA AAGGCATGTA CCACCACACC TTCGGGTGTG 24661 GTGATGCACA GCTTTAATCC CAGCACTCAG GCAGGCGAAT CTCTCTGAGT TTGAGGCTAG 24721 CCTAGTCTTC AGAGTGAGTT CCAGAACAGC CAAGGCTACA CAGAGACACT TTGTTTCGAA 24781 AAACAAACAA AAACAAAAGA GGCTAGCCTG AAACTCCTGA TTCTACCAGC ACCTCCCAAG 24841 GGCTGGGATG ACAGGTTGTG GCCCCATGCT CTCTGCCGGG GCCTCTCTTT TCTTTCTTCT 24901 GTTTGAGGTA GAGGCTTACT AGGTTGGCTG GGTGAGTTGT GAACTCACTC TGCAGCCCAC 24961 ACAGGAACTG ATCTTGTGAT CCTCCTGCCT CAGTCTCCCT AGCAGCTAGG ATTGCAGGCC 25021 TGCACCATCA GGCCCATCGT ACACTGTTTT CTGAGTTTGA AAATTGCCTC TGTTGTTGAC 25081 TATAAGGCAT GCTCTCCTCC TAACATTGTC CTTGGTGCCT CTGCCACCCT TTGGGACTAG 25141 AGAGAACAGA TCTTATTCCT ATTTCACATG CTGTGCCAAC CCAGTAACAA ACTCAGATTC 25201 CTGCTTCCGC CCCCACCACC CCCATCTAAT TGTTCAGTGT TTCTGTGAAG ATAAACACGA 25261 TCATCTTTGT GAAAGCCACT TAAGTTCCTT TCAAGGTTGG GATATAAGTT AGAGTGATAG 25321 CTTGTTCCCA GGGTGGGGAG AGCATGTGAA TTCCCCTCTC GCTCAAGTAG GCTATACTAA 25381 TTTTCATTTA GATATTTCTG AGGCAAAGTC TCATGCTGGC CATCCACCTG CCTTAGCTTC 25441 TCAAGTGCTT GGATTACAGG CATGAGCTAC AATATCTGGC TTAGTTTCAA GGTTGTGAAA 25501 ATTATACTGT GTTCTGATGA CCTGAGTTCA ATTCCCTGGA CCTGGGTGAT GGACGGAGAG 25561 GACAGACCCC TGCAGATTGT CCTTTGACCT CCCTGTCACT ATGTGAACAC TCGTGTACAC 25621 ACACACACAC ACACACACAC ACACACACTA AATGAATGTA ATAAAATATA AAAAGGTGTT 25681 CACTAGTTAA TAAGACATGA GAGAAAAAGC TTACCATCCC TAATCAATGG GGAAGCATTG 25741 AATATAAGTG ACTGTGGTCA TGGAAAGCAG TATAGAGGTT CCTCAATAAA CTGGAATATA 25801 GGAGCATATA CTTGTAAGCC TCCCACAACA GGAGAAAGGT AAAGAGGGGC GGCCACTCTG 25861 GAATATTATT AATATCCTGT TTCATAAACA AGTAAATAGA ACAAACCCCT CAACAACAAG 25921 AACCGGTGTG CTGGCACACA CCTGCAATCC CAGCATTTGG GACTTGGAGG CAGCACAATT 25981 GAAGTTCGTT CTTGGTCATC CTCAGCTATG TATGAAATCT GAAGCCTGCC TGGCCTACAG 26041 GAGACCCTGT CTCAAAAAAA TAAACTAAAT AGATTAAAAT GAAAATTAGA AGCAGGTAGT 26101 GTGGAAGTTG AATAAGAATA GCCGCCATGG GCTCATGTAT TTGAATGTTT AGTGGCACAA 26161 CTTGAGTGAG TTAGGAGGTG TGGCCTGTTG GAGTTGTGTG TCACTGGGAG TGAGCTTTGG 26221 GATTTTAGAA GCCCAAGCCA GGCCCAGGGA CTTGCTCTCT TCCTGCGATC TGAGGAACTG 26281 GATGTAGAAC GCTTAGCTAC TTCTTCAGCA CCATGTCTGC CTGCATGCTG CCATGTTCCC 26341 TGTCAAAATG ATAATGGACT GACCCTCTGA AACTTGGTCT CTTTTGGCTG AGGAGTTAGC 26401 AAGGTAAGAG GTGGCTGTGG CTTGCTCTTG TTTCTCTCTC TCTGATCTTT CATCATTTTC 26461 TCCCGTATCT GGCTGTGGGT TTTTATTATT AAGAGTAATT AGAACTCATG TTACAGTGGT 26521 ACATGCATGC CACAGACCCA GTGTGGATGC CAGAGGACAA CATGTGTAAA TTTTTTCTTT 26581 CCTTGTATGT GCGTCCAGGC TAGTTTCAGA CTTGTGGGCT TCTGCTTCAG CCTCCCAAAG 26641 GTGGGGACCA CAGGCTTATA TACCTACACT CACCTCTTTA TTCCCAGTGG ATGTGTGTGT 26701 GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTTTGTGT GTTTTACACA GACCTGTACC 26761 ACATTCATTT GGTTACTTTT TTTTCCTGCA TTTTGTTTTT AGGTAGGGTC TCACTATGTA 26821 ACCCTGACTG TCCTGGAACA TGCTATTTAG ATTAGACTGA CCTGCTGGTC CCTACCTTCC 26881 GAGTGCTGGG ATTAAAGGTG TGTACTACCA TACCTGGTGA TTAGTTTGTC TTTTGAGACT 26941 GGGTCTCTTG TAGCCCAGGT TGGTCTTGAA CTCCTGGTTT TCCAGACTCT ACCTTCCAAA 27001 TATTGATATT GCAGGTGGTC ACTACCATGT GTGGAATTTA TTTTTGAGCA GTGTTCTGTG 27061 GGTGGATGAT AAGGTCATGT CTATGGTAAA ATTGTTTCTA ATAATGATGA ATAGCTTCAT 27121 GTGTGTATGC ATCTATCAGG TTTGTTCAAC CTGAAGTGTA GGCCTAATAT TTGGATTTAT 27181 TTAGCCAGTG ATAGCTATGA ATTGAGCCCA GAAAAAATCA TAAACTTGAC TAAAACATCT 27241 TAAGAATTTT GTAACTTCTT TTGTAACTCA ACTGTATTGT TTCTGAGCAT GAATGTTGTA 27301 AATGACAATG TCAGCTGCCA TGTCAAAAGG TTGAACATTA CTTGGCAGTG GTGGCACACA 27361 CCTTTAACTC CAACACTCAG GAGGCAGAGG CAGGCAGATC TCTGAGTTAG AGGCCAGCCT 27421 GGTCCACATA GGGAGTTCCA CACCAGCTAA GGTGACAGAG TGAGACCTTG TCTAATTTTT 27481 TTTTAAGGTT GGACATGTAT AATTCCAGAG AATAATTTTT CACTAATCGG AAAAGAGGCA 27541 GTTTCAACTT GGAGTTCACA AGATTTAATC TTTCTTTGAA GATTTATTTA TTTTTAGTTA 27601 TGTGTGTGTA TATATGTATG TATGTATGTA TGTATTGGTG TGTTAAACCC CTGGGGCTGG 27661 AATTACAGGT GGTTGTGAAC CTGATGTTGT AATAAGCTCC CAGACCGTAG CACAAATGAC 27721 TCTATGAAGA AAGTACCATT CAGGCTGTAA AATCCACATA GACAGCACCA CCTGGAAAAA 27781 CTAAAACAAA AATCCAATCC ATCAAACTCC ACAGATCTGG GAAAGTATCT AAATGCACTA 27841 ACCTTGATTT TTGGCTTCTG TAGTTCTGCT TCTGGCTAAC TATTCTTGTT AACTGAAGTA 27901 TGTGAACCCA CAACATGGTT TTTGTGCTTA AAAGTTCTCT GTTCTACAGA ATGAATTCCA 27961 GGACAGCCAG AGCTGCATGG AGAAAATCTG CCTCAAAACA AAACAAACAA ATAAAAACCT 28021 TGAGAAAGGC TCAGGGCTAT ACTGGTATCC CATACACTCA GTGTAGTCGC CAACTGTCAA 28081 AGACTTTTTG TTGACTTAAA CCCATTTCTA AGCAGTATTC TCTTATGGAT ACCCCTTACA 28141 AGTGGGTGCT GGGACTTGAA CTCAGGTCCT CTGGAAAAGC AGAGGATTTC TCACCTGCTG 28201 AGCACCTCTC CAGGCCCATA AGATCTATCT TAAGACAAGA CCTGAGCAGC CTTATGGAGA 28261 TGGCAGTCTG GGGAACCACT GGTGCGCCTT TTCTTCTGCT GGTCACAAAC TGCTGTGGGA 28321 ATTTCCATCT GAAGTTCCTG CCTCTTCTCA CATTCCATGA TATGAGAAAG CTATCAATGT 28381 TCTAAATCTG TTTGCTTTCT GCTTTGCAAG ACCTTTCTCT TTCCTAGGTC ACCCTCCAAG 28441 AGTTCTTGAC CTCAGCCCCG ACTGGTGTCT TGGGATGGGT GACTGGGTTC TGGGGGCTTC 28501 CCTGTGCCTT GGAATATGGT AAAAGAGCAT CTCAGGTATT CACTCAGTAG ATGCTAGTAG 28561 CACTCCCTCC CTCCATTTCT GTCTACAGAT GTTGCTAGCT GGCCCCTATG AGGTAGTCTT 28621 TGCCCCTTTG TTATTGCTGC AGACTCAGAA AAAAGAGGAA ATATAGAACT CCTCGTGGTC 28681 TTCTACTCAA TATCCAAGCA AGGGGGAACA ACTGAGCATC CATACACTGC TGTTTTGGCT 28741 TCTCAATTGC TTGCTTGTAC ATCACCAAGA AGCTTTCATT GGTCAGTGTA AACAAGATCT 28801 GGGAGTTGAT GGTAGAGCAG TTGGATGAGT GACTCTGTCT TTCACCTTTG TTGAGTCATT 28861 TGGTGTGTGC ACATTGTGGG TCCCTGCCTC GCTTCCCATT AAATGTCAAG GTGAACTTTA 28921 TGAGGTTGAA ACTTTTATAT GTAGTGCAAC TGTACTCCTT CCTCTCTATC TCTTCCTTCA 28981 TTTTTCTTCC TTCACCTTCT CTTCCTTTAA AAAAAGAAAA ACTTTAAAAA ATGTGAATCT 29041 GATGTATCCC AGGATGGCCT CAAACTGTTT GCTTTCTCAG AAGATGACCT TGAACTTTCA 29101 ATCCTCCTGC CTCCACCTCC CAAATGCTGG GCTTACAGGA ATTCATCACC ATGCCTGGTT 29161 TTCCTCTCTC CTGGTGAGTG AATCCAGGGC TTCATGCTTG CCAGGCAAGT GTTCTGCTGA 29221 CTGAGTTACA TGCTTAGCCT GTATCCACAT CTTGAGTGAG TAATTTCTGC ACCAAAACTT 29281 TAGGTTTCAT CTCAGTGACT CTGCCAATGT GTTTCCATTT TAGAGTGACG ACTGGCCTTA 29341 GAGGAGAGTG TAAGAGAAAT AGAGTCTCTT TCCTTGGTCT GCTTTTTAAA TTTTAATTTC 29401 TTTTTAGACA TCTTATATTT ATTCATGCAT GTGTGTGTAT AACTAGCAGA ACTCAGCTGT 29461 CTCTTTCTAC CACTCAGGTC ACCAGGCTTG GTGGCAGGGA CTCTTACCTG CCTTCGAGCA 29521 GGCTCTGCCC TCCTTTTGGA GAAACTGGTT TGCAGAAGGA AGAGACAGCA CAGCTCAGAA 29581 GACAGCCGTG CTTTCAGATG CCTGAGAATC CTGCCAAGGA CACTGCTGCA TTCTCCTATT 29641 CTTTTGTAAG GGTCCCATCT CTGCTGAGCT AAACTGGGCT TTCTCAGCCC TTCTCCTCTG 29701 ACAGTATTTT AAAACCCTAC CTAAAGGGGG ATGGAGAGAT GGCTCAGCAA TTAGGAGCAT 29761 ATCCTACTCT TCCGGAGACC CCTACTTCTG TTCCCAGCAC CAATGCTGGT CAATTTACAA 29821 CTGTAACTCT GCTCCAGGTC ATCGGATGCT GCTATCCTCC TCAGGCAACT TCACTCATGT 29881 GCACATACAC ATACTTAAAA ACAAAATAAG TCTTTAAAAA TCACCTAAGA AATATAAAGG 29941 CACATATCAT AATTCAGCCT GCTGTGACGT ATAGCTATAG TCCCAGAATT CTGAAGGCAG 30001 AGGCAAGAGG ATCACCTCAA GCTTGGGGCC AGCGTGGTCT ACAGTGAGAC CCTGGAGACT 30061 TTAATCTCAA AATATGTAAC AAAACAAATA TGTAAATAGA CATATATCAC AATTTATATT 30121 TAAGTAAAAT GGGGGGCATT GGAGAGATAG CTTTGTGGTT AAGAGCATGT ACTGTTCTTG 30181 TCAAGGACCC AAGTTTGATT CCCAGTGTCT ACACTGGTTG GTCTCCAACC CAATTCCAAG 30241 AGATCTGCTG CCTTCTTCTC CTCTCTACTG GAACTGCATT CATGTGCAAA TGTCCATATG 30301 CACACACATA CCCACATGCA TACACACAAA CACATACATA CTCATTTTGC CTGACATCGT 30361 GGTAAAGTGG GAAGACTTGT TGCCCTATTA CTTGGTCTTC ATTTGCCTAT GAGCACCATG 30421 TTGGCATGAA CTCATTCATT AATATCTTTC CTGTACAACT CCCCAATAAC CAAGATGACA 30481 CTTGGCACAC ATTAATTGCT AAGTATAATG AAAATTTAGT TTAAATTAGC TAAATAATTT 30541 AAAGTTCCCC CTCAAGCCTC ATGCCTGATT TAAAGTAGTA CTTATTAATG CTGGGCCTGG 30601 TGGCATACAT TTCTAATTCT AACACTTAGG AGGCTGAGGC AGGAGGATGG CCAATTCAAG 30661 GCCAGCTTAG CCAGCTTAGT AAGACCTTGT CTCCAAGCAA ATTACAGCAA AGTCTGAGAT 30721 ATAGTTCAGT AATTAGGGTG TTTGTCTACC ATGTGTGAAG ACCTGAGTTC AGTTTCTAAC 30781 AACAAAACAA AACTAAACAA ACCAGAACCT AGAGGTTATC ATTTATTTTT TTATTTTTAT 30841 TTTTTTTTGG AGTTTATGCC TTTGGATTAT CCATTCTATG TCCAGACATC AGTACTGCCA 30901 TGTTACAGTC AATAAAAGTC TTCCTTCATC ACCCTTAATC TTATCACCAC TAAAGTCTCT 30961 ACTTGACAGA CATGCCATAC ATAATTATAG CTGTTACCTT CTATCATAAA GTAGACATTT 31021 TATTTTATTT GTGTATTCAT TTTCATTTAT TTTGTTGTTG TTGTTGTTTT ATGAGACAGA 31081 GTTTCTCTGT GCAGCCCTGG TTATCCTGGA ACTCACTCTG CAGACCAGGC TGGCTTCAAA 31141 CACACAGAGA TCCACCTGCC TCTGCCTCCT GAGTGCTAAG ATTAAAGGAG TGTGCTGCCA 31201 TCTTCCCAGC AACATTCTAA ATTATTTTTT GTTTATGTTT TGAAATGGTC TAATGTAGCT 31261 GAGGTGGGCC TCAAGCTTGT TATATAGCTG GGGAACCTTG AACTTGTGTT CTTCCTACCT 31321 CTAGAACTCT GGAGTGCTGG AATTACAGGT ATGAACCATC ACATTCCAGT TTTAATCAAA 31381 TCCAGACTTC ATGGGTACTA GGAAAGCACT CTACAAATTA AACTTCACCC CTAGTTCATA 31441 TATATATATG TGTGTGTGTG TCCATGTATG TATGCCTACA TGATTTTATG TGTGCCACAT 31501 GTGTGCAGGT GCTCTTGGAG GTCAGAGGGT GTCAAATCCC CTGGCACCTG AGTTATAGGT 31561 GGTTGTGAGC CACCTGATGT GGATTCTGGG AACTGAACTT TGGTCCTCTG CAGGAGAAGT 31621 CACTGTTCCT CTGAGTGAAC GTTTCTACTT TTTAATATAC TTCCCATTCG AATTAGAAAG 31681 TAGAAGCTCT CGGAGGTTGA GACCTTACCT AAAGTCACCC AACTAGTAAG AAAACTAAAA 31741 TATCAACTTG GTTTTCTGAG TTTTAAATAT TTTTTCCCAA TGTGTAATTA CACAGGAGAA 31801 TTAATGGGGA CACTTCAAGG TAAAACAGAA GCTTTAGACA TAGCAAGGCA TGGTGGCACA 31861 CATCCCATTG AGAGGCAGGA GGATCAGGAG GCCAGCTTTG GCTGCATACT TAAGAGGCAT 31921 CCAGGGCTAC ATGAGGCGCT ACCTAAAAAA ATTAAATTAG GCAGGGCGTT GGTGGCGCAC 31981 GCCTTTAATC CCAGCACTCG GGAGGCAGAG GCAGGCGGAT CTCTGTGAGT TCAAGGCTAG 32041 CCTGGTCTTC AGAGCGAGTG CCAGGATAGG CTCCAAAGCT ACACAGAGAA ACCCTGTCTT 32101 GAAAAACCAA AAAAGCACTG GTCATTGTCA TTTTCTTTCC TAACAGGGCA CTGGAACCCT 32161 GATGTTGGTT GGCTCCTAGA TTTCTTCTCC ACAGCAGAGA GTTCTTGCCC TGTTAGAGCC 32221 AGAAGGATGC TCTGGAGAGT CAGTATATAG CAAAGCAGGG TCATCTGGAG TAGTAAAAAC 32281 CCTCTGGCAC AGTCAGACCT CATTTCCTCT TGTCCTGTGC TCGTGGCTCT AGCATTATGC 32341 AAGGAGAGGC GCAAACAGCA AACAATTTGG AAGGGCTAGC ACTTGAGCAA CTCTTTGTAG 32401 CTTCCTCTTC TCTACTCTTT TGCCCCTGGC TTCTACTGGA ACAGGTGACT TTCCATTGCA 32461 TTGCATTCTC CAAACTCAGA TGATTTTGAG AATGTGGCAC TACTAAAAGT CACATGGACA 32521 TACAAGGTAC AACTAGAACT ATCCCGGGAA ACAGTGATAC ACGATCTAGT TTGAGGCCTT 32581 GAGCCATAGC TTGTCAGAAG CTCAGAAATG ATTGAGTCTC TGGGAGCCCT CACCTCAGCA 32641 TCCCTGCTTG CAAAAGGCTT CTTGAAGTAG TAAAAACTGC TGGGACCTTG TCTAGGCTGG 32701 GTAACCTTGC ATAATTACTC AACCTTACTG AGCTCAGTCC CCTCCTCTAT AAAATAAGTG 32761 CAACAGTATT TACCTTAGTG GCCCACCTGA AAACATCACA GCTGCCATAG CTAGCTCTTG 32821 GCTTTTGTTC TATCTCCTCC TCCCCCTACT TTCTCTTCCC TCCCTCCCTC CCTCCCTCAT 32881 TTTTCTTTAT TCCTTTCTTT GTATTTTTTT CTTTTTTCTT CCTCACACCT CTCCTTATTC 32941 CCCACCCTCC TCTCTCTCTC TCCCTTCCCA CTTCTCTTTC TTTCATGGCA GGATATCATG 33001 TATCCTAGCT ATACTTGAAT TCACTATATA GCTGAAGAGG AGCTTCCAGC CCTTTTGCCT 33061 CTGCCTCCCA AGTGCTGAGA TTATAGGTGT CCACCTCCAC GTCTACTTAT GCTTTGCTAA 33121 GGATCAAACC AGGGCTTTGT ATGTGCATGC TAGGCAAGAG CCAACTACAT CGCCAGACCT 33181 ATATAATACC CCTTTCTCAG CGAAACTGGG GTTGCTGATG GCTGGTGTTG GGGGAAGGCA 33241 CTAAATATTT AGCAGAAGTA TAGGAAAACT CTAGAAGTCT AGAGATCCTC AAAGTAAGTT 33301 TGGAGAGGCT TGGCCTTTTC TTAGTTGAAA GTCATGGTGC CTACTCACTT TGACTGCTCA 33361 AGGAATATCC ATTCACCACC TGGAAATAAG AAAGGAGGGA GAACCAGCTA GGGATGTGAC 33421 TTAGTAGTAG AGGACTTGTC TAGCATGAGC GTGGTCCTGG GTTCAAGCTC CAGTACAAAG 33481 GCTGGGTGGG GGGGTGGAGA AAGGCTTCTT TCCCATGGCG TTCTAGAGAT GGCGGGGAGA 33541 AACCACCAAT CCACATCTAT CTACAACAGT TCAAGTAGAA CTAATCTTGG TGGTATGGCT 33601 ATAGTAGTCC TAATCCCATC TCAGGGATGC TTCTCTTTGC AATTGATACA AAACACATTA 33661 CAGAAAACCA CAGTGAATCA AAATGCAGAG TTGTGGTGCC TAGTTCCAAT GGATGCATCT 33721 ACAGTACAAC TCCCATGCCT AAGGCTCAGG GATCATTGTG GAAGACAAAG ATCCTCCCAG 33781 GAGATCAGGG AGTTTGCTGT CTCCTAGGAA TTTCAGAAAA TACATCTGTA AAGGCTCACC 33841 AACGTGAATT CCTAAACATG AGCTGAACAA GGATGACAAT AGACATGCTA ACAAGGATGG 33901 GAAAAAGCCC TTGAAGCCTC AGACCTACAC AAAGAGCCGC AGTTGATTAA GGAATGCTGA 33961 TTGTGGGAGA AACCATCTTC CCAAATTGTT ATCTAATACC ACATAGTCAG CCCTGAAAAC 34021 ACACATGCAA ATAAGATTAT ACAAAACAAG GGGGTTGTAC ATATGTATTT AGGAATATAT 34081 ATATATATAT ATATATATAT ATATATATAT GTAACAATAA TTAATAGAAA AAGAGACCAT 34141 GAATTTGAAA AAGAACAAGG AGGGGTACAT GGAAGGGTTT AGGATGCTTT GACCCTTTAA 34201 TATAGTTTCT TGTGTTGTGG TGACCCCAAT CATAAAATTA TTTTTGTTGC TAGTTCACAA 34261 CTGTAATTTT GCTGCTGTTA TGAATTGTAA AGTAAATACC TATGGTTTTT GATGATCTTA 34321 GGCAATCCCT GTTAAACTGT CATTCAGTCC CCAAAGGGGT CAAGACCCAC AGGTTGAGAA 34381 CTGCTGATTT AGAGAGAGGA AAGGGAAGGG GGGGTGAAAT GCTGTAATTA TAATTCCAAA 34441 AAAAAATTTT TAAAAATTTC TTAAAGGAAC TGAAGAAAAG AGCTGAACAT TCTAAGCTTA 34501 AGGGGGGAAA GGTTCTGGAA TGTTACATTT TTCTGGTTTC CTTAGTCTCA GCAACAGGCT 34561 CCCAGCCTTC TGTTTGGACA GTGGTTTACA GGCATGTGAG CTCAGGGAAC ACTCTTCCAA 34621 GTGAATCAGA CTTCAGGAGA AGACATTCAG TTCAGGGCCC TGGGGAAAGT AAGGACAGAA 34681 CTCCATTCCT GAGAATTACC AGGTTTGCTC AGAAGATAAA ACTGGTGAGC CCAATGGCTG 34741 TGTGCACAAC CCTGACCTCA GTGTCTAGGA TAGCTGGACT CTAGCTGCTA GAAGATAGTC 34801 AGAGGGCCAT CCTTTCCCTG AGGCTAATCT GTGAATCAAG TAAACTACAG TCAGGAAGGG 34861 AGCTGGAGAT GGGGGCCCAG CAAACAGGTC CCCCTTAAAG CCCAGCACAT AGGTGGGGAA 34921 CCCAACCTCC CATTTTGTCT TCACCCCACC ACCAGGCCTT TACCAAGGCC CGAGGTTGCC 34981 ACTATTTTCA GCTTGCCAGG CTCTTTGCAG TTTTAGGGGG ATGAGGAGGA GATGCTCTGA 35041 GGTGCTGGGA GGCACATGGC GGGTGCTATT TATGGCTTGG GCTGAACTCC GATGTCCTAG 35101 AAAGAGTGTT TCTGACACTT TCTGCCTTCT GGGAATCAGG AGACTCATGA CAAACACTGC 35161 CTGGCAGTGT TTCTTTCTTG TTCACAGCAA GAAGTGTGCA GTCCATGGCA CGAAAGAGGC 35221 CTGAGGAGGG CAAGATGGAC ACGATGACAT CACTGAAGGA GCTTGCCAGG GGCTGTCTTG 35281 ACTGCTTCAT TAACTCATTC ATGCAGTTTA TTCAGCAGCT ATGCCTGTCA GACCCCATTC 35341 TGTCTGCACA AGACACATGG CAACAAAGGA GACTTACTAT TCCCATCTTC ATGGGTTTTA 35401 TGTTCTGGCA AGAGGAAGAT AGTAATAATT TTTAAAAAGT AACCAGTCTT GAGAGCATGA 35461 TAAATATGGT TGATAACAAT ATGCTATATT TTAAAAGTTG TGAGATAGTA TACTTTAAGT 35521 GTTCTCAAAA CAAAATGATG AATATGGGTG ATATAACATG TTAATTGGTT TAATTTAGCC 35581 ATGCCTTTGT GAACATACTG TATCGTGTAT CATAATTGTG CATGACTTTA TTTATGAGCT 35641 AAATAAATGA ATGGAAAAAA AAGTAACCAG TCTTGATGCT TACCTGCCAT CCTGGAAGGA 35701 AATGGAAATA GGATCTGCCG CCGCAGCATT GCCCTATGCT CTTATTTCTT CTCTTGAAGA 35761 GGTAGGGGTG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTTACTAGA GACTGAGCTA 35821 CCGGCCTCAC ACATTCTAGG CAAATGCTCT ACTTTATATT AAACACTTTA TAAAACATTA 35881 AGCCTTTCAG GGTCAGCAAG GTAGCTCAGA GAGTCCGGGC ATTTGCTACC AAGCCTGACA 35941 ACCTGAGTTC GATTGATGAT CCCCCAGACT CACGTGATAG GAGGAAGCTG ACACCTGTGG 36001 GTTGTCCTCT GACTATAGGC ATGCACACAC ATCCCATGAA TAAATATTTA TACATTTTCA 36061 AATCATACTT ATTTTACAAT GATTTTTATT TGTTTGCCTG TCTTTCTGTC TGTGTAGAGA 36121 CAAGGTTTCA TGCAGCTCAG GTTGGCCTCA AACTCACTCT GTGGCAAGGA TGCCTTAACT 36181 TCAGGTCTTC CAGGTCCAGG TAACAAAATG TTCAGGAGGA ACCTGGTACC TCATCATAAC 36241 CGGTTCTAGA TGGTCTTCCC AGGGCTGCTG TAAGAAAGTG CTACACGACG AGTTATTTCA 36301 AACATTCTCA CAGTTCTGGG GATTAGAAGT TTGAAACTAA GGTGCTGAAG AGATTAGTTC 36361 CTTCTGGAAG CTCAGAAGAG CCATCTGGTC CATACTTTTC TCCAGGTTTC TCTTAGTTTT 36421 TGGCAATCCT TGGAATCCCT TGGTTTGTAG ATGCAGCTTC CAAAGCTCAA GATCTCTCTC 36481 CAATGCTGTG TGGCATTTCC CCGTGTTTAT GAGTGTCTAA ATGGCTTTTA AAAACATTTT 36541 TGAGATGTGA AATTCTGGCT GACCCAGAAT ATATAAACCA GGCTGACCTT TGTCTCCCAG 36601 AGATCTCCCT GCCTCTGCTT CCCAAACCTT TTGATTAAAG GTGTGTGTCA AGTGCCCAGA 36661 CCAAATGCCC TTCTTGTAAG GACAACGGTC ATATTGGATT TAGTGTCTAA GTGAGTCCCC 36721 TATGAACTCA TCTCGAACTC AGTTTGCATA GAACACTGTA CCATGCAAAA TAAATGACAC 36781 AGAGACTGAT ATTGGGGTTC ACACTTCAAG CTGAAGGTCA GAAAAGCAAA GCATTGGGCC 36841 ACTAGCTCTT ACCACTACCT CAGGCTGAAC GGGCTGATCC TGCTGCCTCT CCTCAGCATG 36901 GCTGGAGAAT ATCTTCATAT CCTCATTGTG GCTGGAAAAT GAATGCCTGA TATGGAGAAC 36961 TTGCTCCTGT TTTATATAAC TCCCTAATGC TGGGATTAAA GATGTGTGAT CCCAGGTGCT 37021 GAGATCATCT TTGTGTGAGC TGTTTCTCTT TAGGACTGGA TCAATTTTGT GTAGATCTGG 37081 ATGGCTTTGG GGTCACTGAG ATCTATCTAC CTCTTAATCC CTGGTCCTAG GATTAAAGGT 37141 ATGTACCACC ACATCCTAGC TTCTGGCTGC TGGGATTAAA GGTGTATGCC TGGCTTCGAT 37201 GGCTTGTGGC TGACTTTGCT TTCTGAATCC GCAGGCAAGC TTAAAAAAAT CATAAATAAT 37261 ATATCACCAT AGACCACACT TCCAAATAGG CTTCCATTTA GAGGCGCCAG TGGGTGATAA 37321 TGTAGGCGGT TTTACTCAGT TTTGTGCAGA TGGCTGGCGT CCTGTCTGGT GAGTTCAGAT 37381 TTTTTTTTTT TTTTTTTTTA AGTTCAGAAT CTTACCCAGC TCAGCTTTTC AGGCTGCATT 37441 CAGTGTCCGG CTTTTTTCTC ACCGTCTTGA CTTCCTGTCC TGCATCCCAT TTCTCAGCCT 37501 GGACCCTGCC AGTCTATCAG ATAGATAACA TAAACAAAAT TGTACTGGAT TAATGGGAGC 37561 TGTTTGGACA TTTCCTACTT TTGCCTTTTC ACCAATGATT TGCATACTTA AGCCTGCAAC 37621 TACAGCCCCG ATGCAGTAAG CTCAGTCTCT GGCAAGCAAA GGTCTCTCTG GGGTCTTGTT 37681 TAAGAACCAG CTCAGGCTGC TGGCTCTGTT GGCAGTGGAG GTATTTCCTA TAATGGGATG 37741 ATGGGATGGG TTATTCACAC ACATCTCAGT TACTGGGCTA CATGGATCCA AATCAGCCAC 37801 CCAAGGGTTT GCAGTCACAT GTGAGTCACT TAGCACAGAG AAAGAAGCCT GGAGGAGGAG 37861 GGGTCCTCCC AGCTTCAGGA GGGTTTTCCA GGATATAGGC TTCTAGTCTC GTTTTGGATC 37921 AATTTATCAG TTTTGGATTG GGTCTAATAA CTCTTTCCTG AGCCTGGACT GGGCTCAAAG 37981 GCATGAGTAT GTGAGGGGAA TTTACTAGAA TTCACCTGTA GTTTCTGTAT CATTCCTAGA 38041 GAAGGGGAAG TAGAGACACT GGTGATGGGA AATAAAAACA AAACAAAACC TAAATATTGG 38101 GAGCACAGAG GTCCTTGTTC CACAGCTCTT GATAGAAGTC AGGAATGTTA TGTATGTACA 38161 ATTGCCCTTG AAAAGGAAAG GATGTATGAC CTGTTTTTCT GTCCCGAAGG CTGGGAACTG 38221 GGGATGATTA ACAGCCTGTT GATCTGCATT ATCTGAAGGG CTAGGCCATA TCAAGCTCCC 38281 ACAGCTAGCA CTGAAGGAGA ATAGGGCCTT ACAAAGGGAA TTCCCTCTTT GGATCGAACC 38341 TAGGAACATC TTCTGTTTTA CCGCTCTCTC CTTGTTTCAT CTGCAAAGGG AGGAGCTTGG 38401 TAGTGATGTT GAGGCAGGCA CCACTTGTAT TTTTCTAAGC CACAGAGACT GTTTCCCTAC 38461 CTTACAAACA TCCCTGTGCA TCACTGCAGC TCTGTCTCTT ATGGCAGTGT CTCAGTTAGG 38521 GCTTCTATTG CTGCGACTAA ACACCATGAC CAAAAAAGCT CACACTTCCA TACTCCTGTT 38581 CATTATTGAA GAATGTCAGG ACTGGAGCGC AAACAGGGCA GGGTCCTGGA GGCAGGAGCT 38641 GATGCAGAGG TCATGGAGGA AGGCTGCTTA CTGGCTTGCT CTCCATGGCT TGCTCAGCCT 38701 GCTTTCTTAT AGAACCCAGG ACCACCTGCC CAGGGATGAC ACCACCTACA ATGGGCTGGG 38761 CGCTAATATG AGGGATCAAA GAGATGGAGT TGTGGGAGGG ACAGAGGGGG AGAGCAATGA 38821 AAGAGATAAT CTTGATAGAG GGAGCCGTTA TGGGGTTAGG GAGAAACCTG GTGCTAGAGA 38881 AATTCCCAGG AATCCACAAG GAAGACCCCA GCTAAGACTC CTAGCAATAA TGAAGAGGAT 38941 GTCTGAACGG GTCTTCCCCT TTAATCAGAT TAGTGACTAC CCTAATTGTC ATCACAGAAC 39001 CTACATCCAG TAACTGATGG AAGCAGATGC AGTGATCCAC AGCCAAGCAC TGGGCTGAGC 39061 TTCGGGAGTT CAGTTGAAGA GAGAAGGGAT CATGTGAGCA AGGGGGTGGG GGAAGTCAAG 39121 ATCATGATGG GGAAAACCAC AGAGACAGCT GACCCGAGCT AGTGGGAGCT CATGGACTAT 39181 GAAACGCCAG ACGTTGTAGA CTCCCTAAGG AAGGCCTTAC CCCCTCTGAA GAGTGGATGG 39241 GGGGTGGGAA GTGGGGACGC TGGGGGACAG GAGAAAGGGA GGGAGGGGGA ACTGGGTTGG 39301 TTTGTAAAAT GAAAAAATAG ATTTTTTTTA AATAAAAAAA GAAAGTGCTT TACATCTGGA 39361 TTTCATGGAG GCATTTTCTT AACTGAAGCT CCTTCCTCTC TGGCGACTCT AGTTTGTGTC 39421 AAGTTAACAC AGAACCAGCC AGTACAGGCA GCAGAAATAC CTTGCAGAAA TATCTTAGTT 39481 CAGGAGTCCA CGGTGGTCTC AGTCACTTCC TCATGTGCCA CCTGAGTTTA ACATTCCCCA 39541 AAACTTGGAA CACAGGCCAC CACATCATGG AGCCCTGGCT TAAAGCTCAA GTTTTATGGT 39601 ATTTTCTTTT ATCACTGTCT ATAATTCCTA AACATGCTAC AATGTTGTGA GCCCTCACCG 39661 TCTCCTAGGT CCATAGTGAC TTCCTGGCAT TAATAGACTG TGCCCCAAGA GCTCTATGGC 39721 CACGACCACC ACCTGCCATT CCCCTCCCCC TCCATGGTCC CAGCCTCACT TCTTCACTTC 39781 CTGGTCCTTC CGAGCCCAAT GTGCAAACCC ACAGAATCTG TCTGCTTATG TAAGTTTCCT 39841 GGTCACTGAG TGGGGTGACT CAGCACCAAG GTGGTGCCCT GCGATTTCCC AGCCCCAGGC 39901 AGGAGAACAA CTGAAATGGA AAACAAGTCC CGTTAATAGG GTCCAGCTGA GAGCCTCCCT 39961 TTCTCAGGGA GTCTGGCAAA TCTACTCCTC GGGGAACTGC CCTGGGCAGT GGAATTCTCC 40021 AGCTCCCTGC TCATTTCCTA GTTCCTCTTC CCTCTTCTCA CCTTTGGCTG AGGATCAGAA 40081 AGGTTCCCAC TGAGGTCTGC TTTGCCCTGG GCCTGCTCTT TTCAGAGTCC CATTTTTGGA 40141 ATGAATTTTT TTTGTCTCCT ACTTTCAAGT TCACATATTG AAGCCATTAT TGCCAAGGTG 40201 ATGGTATCAG AAGGAGGGAC CTTTGGGAGA TGAATGGATG GATTCCAAGA GGTTATGTGG 40261 GCAGAGCACC CATGATGGGG TTGGTGCCTT CATAGGAAGA AGACACAGTA GAAGGGAAAG 40321 AGATGCCGAC TGAAAAACAG GAAGTCTCCT GGAGTAGGCC ACTCAGCCTA TGACACGCCA 40381 GCACTCAGAT CTCGGACTTC CCATCTCCCA AATGGTGATA AACAAATGCT GTTGTCCAGG 40441 CTGCACAGTC TACGGCATTT TGTTGCAAGG GCCTGGACCA ACCAGGCTCA GGCAGGAAGT 40501 GAATCTAGTG TGGGAGGATG TACAGACTGC CACTCAGTCT GGACACAAAC TGTCCTCAGG 40561 GATGAGCTGA GCCACATCTA CCTAAGAATG GCTATTCTTT CCATTTGTTA ACATCAAATG 40621 CCAAGCCCCT ACTGTATGTA GGCTCTTGCT AGCAGTGGAT ATGATGCTAT GTGAGATGGG 40681 AGCAATCCTC TCTGCACAGA ACTATACATA GAACTATGCA TAGAAGACCA ACAGGGAGAC 40741 ATCAGATAAC TATTAACTGT GATAGCTCTG TGGGAGACAA AGAGAATGAG GGAATGGACA 40801 ATGACTTTGA GGAAAAACTA TGATTGAAAA TACTCTATCT GGCTGGGCGG TGGTGGCGCA 40861 TGCCTTTAAT CCCAGCACTT GGGAGGCAGA GGCAGGTAGA TCTCTGTGAG TTCGAGACCA 40921 GCCTGGTCTA TAAGAGCTAG TTCCAGGACA GCCTCCAAAG CCACAGAGAA ACCCTGTCTC 40981 AAAAAAAACA AAACAAACAC ACAAAAAAGA AAATATTCTG TGAGGTAAAC AAGCATCTGG 41041 AAGGGTTGGG AGATAATGCA GGCAAAAATG CATTAGACAG CACACAGTAC AACACAGCAA 41101 TCAAACTTAA TATAAACACA GCAAATGTCA TCTTTGGGCT TTGCCCCATT TCCTGATCTG 41161 ACCATAACAG CCTAGTGTCT GGAAAGCACA CTAAAGCCAT TTACGTCACA CAGGAGTTCA 41221 ATGTTGAGTT CAGAGGGAGG GGGTGGAGGG CAGATTAGCG AGGTACAAGT TCTGGTCCCT 41281 TTGATGAAGT GTTGATGTAC CCATCGACAC CACACAAATA TACCATCATG CTCCATGTTA 41341 GGGTCAGTGA AGGATTGCAT ATGTGACGGT GGCCCACTGG GCTGAGAAAG CCCTATTGCT 41401 TAGTGACATC TGTGATAATG ACATGCGAGC CCTATTGCTT AGTGACATCA CTCTTCTCAT 41461 AGTGTGGGAT CCAATGTGTT TCTTGTACAC TTGTGATAAT GACATGCAAA CAAGTCTATT 41521 GTGCGGCCAG TCACACAAAA AATATATTAT GTGCAGTCAG GAACAGTCCA TAGTACTTGA 41581 TTGGGACAGC ACAAGTCTGT GTTGCTGGTT CACACATTAA TCATTACCAC TGTTTTAGTG 41641 TGCTCCTATA TATATATATT TAAAAATTAC TATAAAATGA TACACCGTGC TGAGCAATAG 41701 CACCTCTTAT ACCTTGTGTT TACTGGATGT ACTCAAGCTA TTTTCTCTTG TGCTTGATTT 41761 ATTTGTATTT GTATTTTTGA GAGAACCTGA TCTAGTCCAT GCTGGCTTCA AACTTGTTAT 41821 AAAGCTGAGG ATGGCTTCGA ACTCCTGATC CCCCAGCCTC TGCCTCCCAA ATGATGAGAT 41881 TACAGGCATA TGCTACCAAA CATGACTTTT ATTTATTTTT ATTACTTAGG TGGTATGGGT 41941 GGTTTGAATG AGACTGTCCC CTTTGGCTTA TATATTTGTA GGTGGACCTT TGGAAAGGTT 42001 TAACAGGTAT GACCATAGTG GAGGCAGTGT GTCAGTAGGG GAGGTCTTTG GGGAACCCAA 42061 TACTCAATCA ATTCCAAGTT AGGGCTGTCT GTCTGTCTGT CCCCTGATTG TGTCACAAGG 42121 CAGAAACTCT CAGCTACTGC TCTAGTTCTA TGCCTACCCA CCTGTTGCCA TGGTCCCTGC 42181 CATGATGGTC ATGTACTTCA ACCCTTTGGA TAGGTGGCCC CCAAATTAAA TGGTTTCTTT 42241 TATAAGTTGC CTTGGTCATG GTGTTTTGTC ATGGCGATAA GAAAGTGACT GAGACAGGTT 42301 TGTTGCTGTT GTTACAAGGT TTAGTCCAGG CATCTGGCAC CACCTCTGGC CTGTGCTTGA 42361 TTCAATCATG TTACCTTTAG AAATAGCAGG CTAAAGGACA TATACCTGTG TACGTATATG 42421 TGTACGTATA TATTAGCTGT ATAGTCTAAG TGTGCACCTG ACTCTAATAT CTAGGTTTGT 42481 GTAAGTAGAC TCCACCAAGC TCACTAAGCA ATGGTATCAC AGTTTTCAGA TAGTGTTCAG 42541 CGATGCTTGG CTGAGTGTTA GTTCTTTTTT TAATATTTTA TTTATTTATT ATGTATACAA 42601 CATTCTGCTT CCATGTATCT CTGCACACCA GAAGAGGACA CCAAATCTCA TAACGGATGG 42661 TTTTGAGCCA CCATGTGGTT GCTGGGAATT GAACTCAGGA CCTCTGGAAG AGCAGTCGGT 42721 GCTCTTAACC TCTGAGCCAT CTCTCCAGCC CCTGAGTGTT TTTAAATCAA GGAAAAAAGC 42781 CTGAGGGAAG GGAGCTCAGG CTGAAGGGGA GGAGTCAAGA CAGTCTGACC CCAAGGCATT 42841 GTGGGACGTA AAGAGTTCTG GGACAAGACT GAGGTCTCTT CCTTCTCAGA GACTGTGGGC 42901 TTCAGTTTCC TTGGTAGCCG GAAGCAAAGC TAATCCATGG CTTAAAATAT AATACTCAGT 42961 GTAACCTTGT GTTGTAGAAG TGACTTGCTT GTCTTCTTCC ATAATTCTAA AACATCTTTA 43021 AGAGCAGGAT CCAGGAAGGG AAAAGGAGAG ATTCTCATCT TCTTCAAAAG GCAGCTTTCC 43081 CTAAAGCATT TTCTGATGAA ATTTAAGTTC TAAAACCAGC AGTGGTATAA TCCCATCATG 43141 AATGGGGATC TCTGAGTTTA AGGCCAGCCT GGTCTACAGA GCAAGTTCCA GGACAGCCAC 43201 GGTTACACAA AGAAATCCTG TCTTAAAACA AAACAAAACC CAAAACAAAC ATAAACAAAA 43261 ACTATCCAAA ACCAACCAAC CCCCCCAACT CAGAAAGAAA GAAAGAAAGA AATCAAGAAA 43321 GAACTGCCCA CCGGGTGTTG GTGGTGCAAG CCTTTAATCC CAGCACTCGG GAGGCAGAGG 43381 CAGGCAGATC TCTGTGAGTT TGAGGCCAAC CTGTTCTCCA GAAAGAGTGC CAGGATAGGC 43441 TCCAAAGCTA CACAGAGAAA CCCTGTCTTG AAAAAAGAAA AGAAAGAACT ACCCATGACC 43501 AAACAGTTCC ATGGCCAGGT AGAGAATGAG GACGCTGAAA GTCACACCTT CTCAGAGTCT 43561 CAAACTGGAC ATCTGGCCTC AAAGTCCAGA AATGAGTGCA AGACCATTAA TGACAGTCTT 43621 TGGAAACAAA CCAGACCAAA GAACATTTGG CTCCTGATAC ATATTCTGAG GGTCACATAG 43681 AAAGAAAGAT CTGCCTTTGG CCACCTCCTT TTGAAGTGGG GAATTTTATT TTCTTCTGCA 43741 TGGAAACTTC ATGTAGGTAT TTGAGAATAC ATACAGACAT GCAGGTGCAC ATGCACGGAC 43801 ATGAACACAC ACATACACCC CGGGTAGGCA GGCAAGAAAG TGTGTGGAAT AACACTTGAA 43861 CTTCCCTTCC AGAACAGAAG CCCTCTGAAG TGTGACATTC ATGCTGGCTG CATGGGGTCT 43921 GATCAGTACT AGTGAGTGGA GGTGGAGGGG TAGGAAACAT GGGGATGATA ATAGGTTGTC 43981 AGGAAAGTGG TGCCCCAGGT AGCACAGAGT AGAAATTTGT CCCCCAAAAT CCTTTTGAAC 44041 CCAGTTGATT TGAATGCCGT GCCCCTGCCA CCCAGGCTTC AGAGCTAAGT GACTTATGTC 44101 TTCAGGTCAG TGATGATTAC CACGGTTGCA GTGCTAACAC AGATGCTTTA TCTACCAGGA 44161 CAGAAACAAG AAAGATGCTC CTTCCCAGGC CCCTTAGCAC TCTCTGGGTG GGGAGGATTG 44221 CCCCACCTTC CAAAAATAGA ATACTGTTTT GGTAAACAGC CACTTTGAGC CCATGAGGAT 44281 ATCTTCATTA GCTATGGAGA CAGGTTTTAG TAAGAAAGCA AGATGAGAGG CTAAAAAACC 44341 CTTGGGGAGC AGGAACTGGG AAGACTGTGG TACCTTGTTC CCAGATCCAC CAGAAACCTT 44401 GCCACCAGAC GATGTGTCCA GGCCCCACAT ATTTCACAAA AAGTTGGATC TGATAACAAT 44461 GAGGATGGAA TCCCGGTCTT AAGGTGGGTT TGGGGTGGGA AGAGGCGGGA TAATGGGTGA 44521 GAGGGTCGGT GGGGACAGGT GAGATGGGGT ATGGTGGGGA GAGGTGGAAT GGGGTGGGGT 44581 GGGTTGAGAT GGAGTATGGT ACAGCGGGGA GGGATAGAAT TGTCTTTTCC CTGTACCACA 44641 GAGAAGTTTG ACTGCTACCC TTGGCAATTA ATCAATTATA GAAAATGCAA CTTTGCTTTT 44701 AAAATGTGTC TATTTCCAAA GGCTTCTTCC CCTCCCCTAC CTAGGGAGAA GGAAAGAATG 44761 GATAATGCTA CTGTAGAGGA GGGTAGCATC ACTATAGAGG CCTCAGTATC TGCCCCAGGG 44821 AGCTGGGAGA GAGTTCTATC ACACAAACAC AGCCCGAGTC ACATACTCAA CAAACCCCAC 44881 AAAACAAAAC AACAATAATG AAGATACAAA ATCTCATTAT GTAGCCCAGG CTAGTCCTAG 44941 ATTTCTGTTT TCTTTTTTTG TTTTTCGAGA CAGGGTTTCT CTGTGTAGCT TTGGAGCCTA 45001 TCCTGGCACT TGCTCTGAAG CCCAGGCTGC CCTCACTCAC AGAGATCCGC CTGCCTCTGT 45061 CTCCAGAGTG CTGGGATTAA AGGCGTGCAC CACTAATGCC TGGCTAGTCC TAGATTTTTT 45121 TATCCTCCTG CCTCAGGCTC CCAACTGTTG GGTTTACTTT TGGGAGTCCA TTTTCTTCCA 45181 GCATGGATTC TTTGAATTGA AATTCAGATT ATCAGGTTTC TGTAGCAATC CCACCAGCCC 45241 ATTTTTTTGT CTGACACTGC TTGTTTTGAG ACACAGTCTC CCACTGCTGT AGCCCAGGCT 45301 GCCCTAGATT TTCTATGTAG CCCAGGCTGG CCTTGAACTC CCAGGAGTCC TCTGGCCTCT 45361 CCCTTTTGAT TACTGGAACT AGAAGAAGTC ACTATGCTTG ACTTGGAACT AATATTAGAA 45421 CAAAATATAT TTTTCATTGA GATTCAACTT TGAAATCCTG ATGCTCCTGC CTCACTCAGG 45481 TCATCAGGGT TGGCAGCAAG AGCCTTTATC CACTGAGTCA TATTGGGCCC TGACCTGCTT 45541 TTAAATTTTG CCTTTAGGGC TGGAGATGTA GCTCGGCTGG TTCAGTGCTT GCCTGGTACC 45601 CACGAAGCCC TGGGTTTGAT CTACAACACA GTATAAGCCA GGCCTGATGG CGTATACATG 45661 TAATCCTAAC ACTTGGGGAG CAAGAGGGAG GCCAAAGCCA TCCTCTGCTA CTTGGTGAGC 45721 TTGAGGCCAG CCTGGGATCC TTGAGACCCT GTTTCAAAAC AATAACAACA AACACAGACT 45781 ACTAAAAAAA ATTAATAAGG GCCAGACTGG GTGGTGTATT CCTTTAATCC AAGCAATGAG 45841 GAGGCAGAGG CAGGCAAATG TCTGTGAGTC TGGGGACAGC CTGGTCTACT GAGCAGCAGG 45901 CCAACTAAGG CTACATAGTG AGACTATCTC AAAAAAAGCA AAATAACAAT AAACAGACCA 45961 GTTCCCCATC TCCTATTTTG CCTTTACCTC CTATTCCCTG CTCAGCAGGT TATTTTTTGT 46021 TCCTGCATCT TGGTTCACTG ATCTGTAAAC TTGTCTGAAT AAGTAGGTAC AGGGTTGTTT 46081 TAAAATTAGA TAATATATTC AATGAGAAGG GCTACCAAGT GCTCAACCAA TGTATGCATA 46141 TGTATGTATG TATGTATGTA TGTATGTATT TATTTTTGTT TTGTTTTTCA AGATAAGGTT 46201 TCTCTGTGTA GTTTTAGAGT CTGTCCTAGA ACTTGCTCTG AAGAGCAGGC TGGTCTTGAA 46261 CTCACAAAGA TCCACCTGTC TCTGCCTCCC AAGTGCTGGG ATTAAAGGCA TGTGACACCA 46321 CCCCCAAAGC CAATGTTCTT ATAGGCATCT TTGATTTTTT TTCTCTTTCT TTGAGTGGAG 46381 TCTGACTAAG TAGCCCATAC TAGCTCTGCA TTTACAATCT GAACACATGG ATAAGAGTGG 46441 TGAAAATTAT CAAGATCATG TTATGCTATG CCTCCTGAGT CACCATGCCC TGCTTCAGAC 46501 TTCTTTGTAT TAAAGAACTG TGTAAAAAAA AAAAAAAAGA CATTTGAAGG CACATAATCA 46561 GAGGAATTTG TCAGTGATTT TTCACATACT GTCTTATTTG TGGCCAAGGT AAGCCTAGAG 46621 AGTATTTCTT AAAATTAAAA ATAGTGGGCA GATTTTGGAG GCGATCTGAT ATGAAAATCC 46681 CTTCCCACCC CAGGTAGTCA TGGGCTGACT ATCAAGGATA CATTCTGAGA CATATATCCT 46741 CAAGGAGTTT CTGCCTTACG CAAATATCAT AGGTCATAGC ACACTGAGAC TATGTGGCAG 46801 TCTATGTGTC TATATACACA TGGTGTGGCC TATTGTTCCC ATGGTCACAA AGAACAAAAC 46861 AACTTTTTCA CAAGGCTTTA CCCCTAGAGG AAGAGCTACA GGCAATCAAT GGTTGCTGAG 46921 AGGAGTATCA GTCTTCTCCA GGGACTTAGC CAATCCCAAG AGGTCAGCCA CGCATAGGAA 46981 CGCTTAGCCA CGCTTGTATA GAACATCTCA AACAACAACC ACCTCAGTGT AAAGCAAGCA 47041 CACAAGGAAC TGATGCAACT AAGAGACAAA GGGCCCGGTG TGTGTGGCCC GTAGCTGTCA 47101 TCCCAGCACT TGAGACTAAG GAAGGAAGGT TGAGAATTTG AGGCCAGCAT GGACTCCACA 47161 GAAAGACCGT TTTCTTTCTC AGAAAAAAGA AGCAAAAACC AAGAACAAGG TGTATGGGAA 47221 TGCTACTGTC TTGGCATATT GTTTATAGAA AACTTTTTTA TATATAAAAG GAATGCACTA 47281 CAAAAATTAT AAACTACTGT AATATTAACT GCATAGATCT ATAACATGGT CATTTATTAT 47341 TGAGTATGAT TATGTATGTA CCCAGGCTGC AGGTTTAGAC AGTTGCACTA CAGTAGATCT 47401 GTTTGCAGTA GCATCATTAT TAGACATTTT GGACAAAGCC AAGTGGTAAT GGCACATGCC 47461 TTTAATCCCA GCACTTGGGA AGCAGAGGTA GGCGGATCTC TGTGAGTCAG AGACCAGCCT 47521 GGTCTACAAA GAACTAGTTC CAGGAGAGTC TCCAAGGCCA CAGAGAAACC CTGTCTCGAA 47581 AAACCAAAAG AAAAAAAGAA AACAAAAAAC TAAAAAATAA ATAAATTTGG GGCAATATCT 47641 TGTCCTATGA TGTTACTGGG TAATGGGATT TCCTCCTCTT GTATTATTTT TTCTTTGGGG 47701 GTTTTACTTA TTATTTACTT GAGACAGAGT CTCATTTATG ACAGGCTGGC CTCAAACAGG 47761 AAATGAAGCC AAGGAAGACC TTGAAGACCT AATCCTTCTG TTTCTTCCTC CTATATGGTG 47821 AGTTAAAGGC ATACAGTACC ATGCCCAGTC TATTCACTGC CCAGGGCTTC ATGCATGCTA 47881 GCAAAGGACC AACTGAGCTG CATCCCCACC CCTCCTCCTG GCTTCCATCT CCTTATGTAG 47941 CTAGAAATGA GCCTGTCTGT CTCAAATACT GGGATTATGG GTGTGTGCCA CCACACCTGG 48001 CTTCCTATTA TAGCCTTGTG GGATCACTGT TGTTTACTGA AGCATTGTGA CACACTGCAG 48061 ATTGCTGGAA CAGCGTCTGC CATCATCATG ACACAACTTC AGAGAAAGAG AGAGTTCCCA 48121 ACCAGCCACA CACTTAACTC AATGCCTGTA GCCCTTATTC TGTTAAGACG ATTTCCTGCC 48181 ATCTTACTCA AAGACCCTCT TTAACTCGGT AGGAACATCT GTTACACTGA AAGTCCTGCC 48241 TGTTGCTCCA CTGACCTCCT TCACAAATTA TTATATTTTG GAGCCAATTC TGAACCCAGG 48301 TTTTCTGAGT GACACATTTT AGTATTTTTT TTTTCTTTCT ATTTTCTTTC ATGGAAAGTC 48361 TCTTGTTACT GTTCACATGA CCAAGGATCA CTGCATCATC TTCCAAGGCC AATTTTGGAT 48421 GTTTCAGCAA GGGAGACTGA AGATCCTGAG TCTCAGTGTT GATCTCCTTT AGAATGTCCT 48481 CTGGAGAAGG TAGTGACAAC ACTGCAAGGA TAATAGGTGA ATAAAGGGAA GCCAGAGTGT 48541 CCTCTGGGAT GTGCGGCACT TACATGAAGG ATTCATTTAT AAATTTTAAG TTATGGAGTA 48601 TAATAATAAG ACTAAATATG TAGTGTCGTA ATTTTATAAC TATACATATG TATATAGTAA 48661 ATATAAATTT ATATGTAATG TATTTATAGT AAGTGTACAT AGAATTGAAC ATATGTTACA 48721 TAAATGGCAG AAAGGAATGA TTCTCAATTG CTTTTTTTCT AATTATAATT TCTATTGCTC 48781 TTTGTGGATT TCACACCATG CATTCTGATC CCACTTATCT CCTTGTCTCC TTGCATTTGC 48841 CCTCTGCCCT TGCAACCTCA CCCCCAAATC AAAGCCAAAT TTAAAAAAAA AACCAAAATC 48901 CAAACAAAAC AGAGACAAAA CAAAAATAAA AGCAACAACA AAAAAAGGAG AATCTTGTCA 48961 TGGTAGCTGT AGTGTGGCCT GTTGAATCAC ACAGTATACC CTTTAGTCCA TTCATCTTTT 49021 CTTCCAAGTG TTCATTGATA CAAGTCACGG TCTGGCTCGA GGATTCTGGT TTCTGCTATA 49081 TTACTAATAA TGGGCTCTCA CTGGGGCTCC CCTTGGATAT CCTATTGTCC TGTGTTATGG 49141 AGAGCCTGCT GTTTTGGATA TGTAGGTTTG TCCCCTTCAC ATGCTATAAC AATTCATAAA 49201 TTCAGTGAAT GTTGGGGTGG GCCAACTCAT AGCCCTGGTT CTGGGCTTGG GTGGTATTAT 49261 TAAACCCACT GATGGAGAAT AAGACCACTA CCATAATTTA AAAGCCAAAT TGAAGCAAGT 49321 TTTAATTCAA TACTGCCCAG GTGGACAGGC TCTGGCTAGG TCCATCTCTG AGTTTCCAGG 49381 AGGTGGCCCT GACTCACGGT TTACAGTGGC TTGAGTATTT TCCATAAGGT CCAATCAGGG 49441 GCAAGCATAC ATCCTGATGT ACCTCCAGTC TATATCCAAT CGGGGGCAAG TGTACATCTT 49501 GATGTATTTC CTGCCTGTGA ACCTACTGCC CACATGTGAT CAAGCACATC CGGTGCAGTT 49561 GGGTCAAACA GACTTGTTTA GGGCAATGAA AAACACATGG CTTTTTATCT CCCATAAACA 49621 ATAGCCTCCA GCGGTTCAGG GACTATTTGT CCTTGGGCAA GGAATTTACA GATCCTATAG 49681 GTGAGTCAGG GTCAGCATCC TGCTCTCATG CCCTCAGGGC TGGCTCACTT GTTACCTCCC 49741 CGACCCTCTC TCAACAGGGT CAGCTCTGAG GTGCTGCCCA GGTGGGGTGC AGGGCCTACT 49801 CTTCCGCATG TTGCAGCTGG TCAGGGTTAG TTCTCTCATA TGCCACAGGT GGCAATGGGT 49861 GAAGGGGGAG GGCATGTTTC CCTCATCAAC GCCATTACAT GGGGGGATGG GGTCAGCTCT 49921 CATGCCCTTA GGGTTGGCTC ACCTGCATCC TTGACCATAG GGTCAGCTCT AGTATGCTGC 49981 TCAAGTGAGG CGCACACCTA (LoxP sequence from bacteriaphage P1) SEQ ID NO: 4     1 ATAACTTCGT ATAGCATACA TTATACGAAG TTAT (FRT sequence from the 2pm plasmid of the bakers yeast Saccharomyces cerevisiae) SEQ ID NO: 5     1 GAAGTTCCTA TTCtctagaa aGTATAGGAA CTTC (attB sequence from E. coli) SEQ ID NO: 6     1 cCTGCTTt t TtatAc tAA CTTGa (Recognition site for the CHO-23/24 meganuclease, 35,699 basepairs downstream of CHO DHFR) SEQ ID NO: 7     1 TAAGGCCTCA TATGAAAATA TA (Recognition site for the CHO-51/52 meganuclease, 15,898 basepairs downstream of CHO DHFR) SEQ ID NO: 8     1 ATAGATGTCT TGCATACTCT AG (CHO-23/24 meganuclease) SEQ ID NO: 9     1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GSIKAQIFPN QCYKFKHQLR LRFQVTQKTQ    61 RRWFLDKLVD EIGVGYVTDR GSVSDYMLSQ IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE   121 QLPSAKESPD KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA   181 ASSASSSPGS GISEALRAGA GSGTGYNKEF LLYLAGFVDG DGSIIAQIKP GQSYKFKHTL   241 QLVFQVTQKT QRRWFLDKLV DEIGVGYVID RGSASDYRLS EIKPLHNFLT QLQPFLKLKQ   301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQ1AALNDSK TRKTTSETVR AVLDSLSEKK   361 KSSP (CHO-51/52 meganuclease) SEQ ID NO: 10     1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GS11AQ1PPN QSCKFKHQLR LTFQVTQKTQ    61 RRWFLDKLVD E1GVGYVRDR GSVSDY1LSE IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE   121 QLPSAKESPD KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA   181 ASSASSSPGS GISEALRAGA GSGTGYNKEF LLYLAGFVDG DGS1YAG1AP NQSCKFKHQL   241 RLWFVVSQKT QRRWFLDKLV DEIGVGYVID NGSVSHYRLS EIKPLHNFLT QLQPFLKLKQ   301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQ1AALNDSK TRKTTSETVR AVLDSLSEKK   361 KSSP (CHO-51/52 donor plasmid with EcoRI site) SEQ ID NO: 11     1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA    61 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG   121 TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC   181 ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC   241 ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT   301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT   361 TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT ACCCAGAAAC   421 CTTTCAACCA GCTTTTGAGC TAATGATAGA GAGAAGCTCA AGGAATTGGA GCAATGCTTG   481 ACTAGGGATG TCAGAGGGAG GCTATCCAGA GGAGCTTACA ACTGAGGTAA ACTTAAAAGT   541 TAGGGAGTTT GTCAACTTCA ACCCACAGAA TAGAGCAGAG CCAGGAGGAG CTGAGGCTTC   601 TGAGTGTTAT GGTGGAAGCA TCACCCCAAC CCTTGACATC CATATGCCTG AAGAGTCTGG   661 AATGTTATGG TGGAAGTTCC ACCCAAGCCT CCCTTCCCGG TCGCCCTCCA AACCCTGCTA   721 CATCTCAGAA ATCCCACCAA ATGATGACTC CCTCCCCCAG AGATATTCAA GACCACTCCC   781 ACAGGGTATT TAAACTGCCC CCCAACCCCC AGAAAATAGA TGTGTGGTTT TCCAATCTCT   841 CTTTCCTATC ACGTCTCTGG GGAGCTGGCA GGCCATTTGG GAGCATTGTA TCCATTAAAC   901 GACTTCTCAG TGGAGACTCT GAAAGCCAGA AGAGCCTAGA CAGATAGATG TCTTGCGAAT   961 TCTTGCATAC TCTAGAGACT ACAGATGCCG GCCCAGACTA TTATATCCAG CAAAAGTTTC  1021 AAACACCATA CAAAGTCAAA TTTAAACAGT ATCTATCTAC AAATCCAATA TTACAGAAGG  1081 TGCTAGTAGG AAAACTCCAA ACTAAGATTA ACTATACCTG TGAAGACACA GGAAATAATC  1141 TCACACTGGC AAAAGAAGAA AAACCTCTCT CTCTCTCTCC TCTCTCTCTC TCTCTCTCTC  1201 TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCACACACAC ACACACACAC ACACACACAC  1261 ACCAACACCA ATACCATGAA CAACAAAATA ACAGGAATTA ACAATAATTG ATGTGTGTGT  1321 ATGTCCCTGT GTGTGTGTCC TTGTGTGTGT CTGTTTGTGT GTCTGTGTAT ATGTTTGTCA  1381 CCTGAGGGGT GGCTCTTCCT TGGTTTGTGA GGTTTCTACC CAAAAGCTTG GCGTAATCAT  1441 GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG  1501 CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG  1561 CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA  1621 TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA  1681 CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG  1741 TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC  1801 AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC  1861 CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC  1921 TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC  1981 TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA  2041 GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC  2101 ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA  2161 ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG  2221 CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA  2281 GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG  2341 GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC  2401 AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT  2461 CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA  2521 GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT  2581 ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA  2641 TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC  2701 GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG  2761 CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG  2821 CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT  2881 CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT  2941 CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT  3001 CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA  3061 AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA  3121 TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT  3181 AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC  3241 ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA  3301 GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT  3361 CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG  3421 CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT  3481 ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT  3541 AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT  3601 AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC  3661 GTC (Recognition site for the CHO-13/14 meganuclease, in Intron 2 of CHO DHFR) SEQ ID NO: 12     1 TACATGTATG TACAAAATAT AT (CHO-13/14 meganuclease) SEQ ID NO: 13     1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GSIFASITPR QCYKFKHELQ LTFWTQKTQ    61 RRWFLDKLVD EIGVGYVIDQ GSVSHYRLSE IKPLHNFLTQ LQPFLKLKQK QANLVLK11E   121 QLPSAKESPD KFLEVCTWVD Q1AALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA   181 ASSASSSPGS GLSEALRAGA GSGTGYNKEF LLYLAGFVDG DGS11AQ1KP NQSCKFKHQL   241 MLTFTVAQKT QRRWFLDKLV DE1GVGYV1D 1GSVSEYRLS Q1KPLHNFLT QLQPFLKLKQ   301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQ1AALNDSK TRKTTSETVR AVLDSLSEKK   361 KSSP (Recognition site for the CGS-5/6 meganuclease, in Exon 4 of CHO GS) SEQ ID NO: 14     1 AAGGCACTCG TGTAAACGGA TA (CGS-5/6 meganuclease) SEQ ID NO: 15     1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GS1KA11RPE QSYKFKHRLR LVFQVTQKTQ    61 RRWFLDKLVD E1GVGYVYDR GSVSDYYLSE IKPLHNFLTQ LQPFLKLKQK QANLVLK11E   121 QLPSAKESPD KFLEVCTWVD Q1AALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA   181 ASSASSSPGS GLSEALRAGA GSGTGYNKEF LLYLAGFVDG DGS1WAR1KP GQSYKFKHTL   241 ELVFQVTQKT QRRW1LDKLV DE1GVGYVTD AGSASVYRLS ElKPLHNFLT QLQPFLKLKQ   301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQ1AALNDSK TRKTTSETVR AVLDSLSEKK   361 KSSP (Forward PCR primer for evaluating CHO-23/24 target site) SEQ ID NO: 16     1 ggagggacat taatctgcat gcagtgatc (Reverse PCR primer for evaluating CHO-23/24 target site) SEQ ID NO: 17     1 gtcttggttt gggttgtcta agcaacctc (Forward PCR primer for evaluating CHO-51/52 target site) SEQ ID NO: 18     1 CACAGGTGTC CACTCCCAGT TCAATTACAG CTCTTAAGG (Reverse PCR primer for evaluating CHO-51/52 target site) SEQ ID NO: 19     1 CGATGGCCCA CTACGTGAAC CATCACC (PCR template for mRNA encoding CHO-23/24) SEQ ID NO: 20     1 CACAGGTGTC CACTCCCAGT TCAATTACAG CTCTTAAGGC TAGAGTACTT AATACGACTC    61 ACTATAGGCT AGCCTCGAGC CGCCACCATG GCACCGAAGA AGAAGCGCAA GGTGCATATG   121 GCACCGAAGA AGAAGCGCAA GGTGCATATG AACACCAAGT ACAACAAGGA GTTCCTGCTC   181 TACCTGGCGG GCTTCGTCGA CGGGGACGGC TCCATCAAGG CCCAGATCTT TCCGAACCAG   241 TGCTACAAGT TCAAGCATCA GCTGAGGCTC CGTTTCCAGG TCACCCAGAA GACACAGCGC   301 CGTTGGTTCC TCGACAAGCT GGTGGACGAG ATCGGGGTGG GCTACGTGAC TGACCGCGGC   361 AGCGTCTCCG ACTACATGCT GAGCCAGATC AAGCCTCTGC ACAACTTCCT GACCCAGCTC   421 CAGCCCTTCC TGAAGCTCAA GCAGAAGCAG GCCAACCTCG TGCTGAAGAT CATCGAGCAG   481 CTGCCCTCCG CCAAGGAATC CCCGGACAAG TTCCTGGAGG TGTGCACGTG GGTGGACGAG   541 ATCGCGGCCC TCAACGACAG CAAGACCCGC AAGACGACCT CGGAGACGGT GCGGGCGGTC   601 CTGGACTCCC TCCCAGGATC CGTGGGAGGT CTATCGCCAT CTCAGGCATC CAGCGCCGCA   661 TCCTCGGCTT CCTCAAGCCC GGGTTCAGGG ATCTCCGAAG CACTCAGAGC TGGAGCAGGT   721 TCCGGCACTG GATACAACAA GGAATTCCTG CTCTACCTGG CGGGCTTCGT GGACGGGGAC   781 GGCTCCATCA TCGCCCAGAT CAAGCCGGGT CAGTCCTACA AGTTCAAGCA TACCCTGCAG   841 CTCGTTTTCC AGGTCACGCA GAAGACACAG CGCCGTTGGA TCCTCGACAA GCTGGTGGAC   901 GAGATCGGGG TGGGCTATGT GATCGACCGC GGCAGCGCCT CCGACTACCG CCTGAGCGAG   961 ATCAAGCCTC TGCACAACTT CCTGACCCAG CTCCAGCCCT TCCTGAAGCT CAAGCAGAAG  1021 CAGGCCAACC TCGTGCTGAA GATCATCGAG CAGCTGCCCT CCGCCAAGGA ATCCCCGGAC  1081 AAGTTCCTGG AGGTGTGCAC CTGGGTGGAC CAGATCGCCG CTCTGAACGA CTCCAAGACC  1141 CGCAAGACCA CTTCCGAGAC CGTCCGCGCC GTTCTAGACA GTCTCTCCGA GAAGAAGAAG  1201 TCGTCCCCCT AGACAGTCTC TCCGAGAAGA AGAAGTCGTC CCCCTAGCGG CCGCTTCGAG  1261 CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA  1321 AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA  1381 ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG GGGGAGATGT  1441 GGGAGGTTTT TTAAAGCAAG TAAAACCTCT ACAAATGTGG TAAAATCGAT AAGATCTTGA  1501 TCCGGGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG  1561 CCTGAATGGC GAATGGACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT  1621 ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC  1681 CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT  1741 TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT  1801 GGTTCACGTA GTGGGCCATC G (PCR template for mRNA encoding CHO-51/52) SEQ ID NO: 21     1 CACAGGTGTC CACTCCCAGT TCAATTACAG CTCTTAAGGC TAGAGTACTT AATACGACTC    61 ACTATAGGCT AGCCTCGAGC CGCCACCATG GCACCGAAGA AGAAGCGCAA GGTGCATatg   121 gCACCGAAGA AGAAGCGCAA GGTGCATATG AACACCAAGT ACAACAAGGA GTTCCTGCTC   181 TACCTGGCGG GCTTCGTGGA CGGGGACGGC TCCATCATCG CCCAGATCCC GCCGAACCAG   241 TCCTGCAAGT TCAAGCATCA GCTGCGCCTC ACCTTCCAGG TCACGCAGAA GACACAGCGC   301 CGTTGGTTCC TCGACAAGCT GGTGGACGAG ATCGGGGTGG GCTACGTGCG CGACCGCGGC   361 AGCGTCTCCG ACTACATCCT GAGCGAGATC AAGCCTCTGC ACAACTTCCT GACCCAGCTC   421 CAGCCCTTCC TGAAGCTCAA GCAGAAGCAG GCCAACCTCG TGCTGAAGAT CATCGAGCAG   481 CTGCCCTCCG CCAAGGAATC CCCGGACAAG TTCCTGGAGG TGTGCACGTG GGTGGACGAG   541 ATCGCCGCTC TGAACGACTC CAAGACCCGC AAGACCACTT CCGAGACTGT CCGCGCCGTT   601 CTAGACAGTC TCCCAGGATC CGTGGGAGGT CTATCGCCAT CTCAGGCATC CAGCGCCGCA   661 TCCTCGGCTT CCTCAAGCCC GGGTTCAGGG ATCTCCGAAG CACTCAGAGC TGGAGCAGGT   721 TCCGGCACTG GATACAACAA GGAATTCCTG CTCTACCTGG CGGGCTTCGT GGACGGGGAC   781 GGCTCCATCT ACGCCGGGAT CGCGCCGAAC CAGTCCTGCA AGTTCAAGCA TCAGCTGCGC   841 CTCTGGTTCG TGGTCAGCCA GAAGACACAG CGCCGTTGGT TCCTCGACAA GCTGGTGGAC   901 GAGATCGGGG TGGGCTACGT GATTGACAAT GGCAGCGTCT CCCATTACCG CCTGAGCGAG   961 ATCAAGCCTC TGCACAACTT CCTGACCCAG CTCCAGCCCT TCCTGAAGCT CAAGCAGAAG  1021 CAGGCCAACC TCGTGCTGAA GATCATCGAG CAGCTGCCCT CCGCCAAGGA ATCCCCGGAC  1081 AAGTTCCTGG AGGTGTGCAC CTGGGTGGAC CAGATCGCCG CTTTGAACGA CTCCAAGACC  1141 CGCAAGACCA CTTCCGAGAC TGTCCGCGCC GTTCTAGACA GTCTCTCCGA GAAGAAGAAG  1201 TCGTCCCCCT AGACAGTCTC TCCGAGAAGA AGAAGTCGTC CCCCTAGCGG CCGCTTCGAG  1261 CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA  1321 AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA  1381 ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG GGGGAGATGT  1441 GGGAGGTTTT TTAAAGCAAG TAAAACCTCT ACAAATGTGG TAAAATCGAT AAGATCTTGA  1501 TCCGGGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG  1561 CCTGAATGGC GAATGGACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT  1621 ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC  1681 CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT  1741 TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT  1801 GGTTCACGTA GTGGGCCATC G (PCR template for mRNA encoding CGS-5/6) SEQ ID NO: 22     1 CACAGGTGTC CACTCCCAGT TCAATTACAG CTCTTAAGGC TAGAGTACTT AATACGACTC    61 ACTATAGGCT AGCCTCGAGC CGCCACCATG GCACCGAAGA AGAAGCGCAA GGTGCATATG   121 GCACCGAAGA AGAAGCGCAA GGTGCATATG AACACCAAGT ACAACAAGGA GTTCCTGCTC   181 TACCTGGCGG GCTTCGTGGA CGGGGACGGC TCCATCAAGG CCATTATCCG GCCAGAGCAG   241 TCCTACAAGT TCAAGCATCG CCTGCGGCTC GTTTTCCAGG TCACGCAGAA GACACAGCGC   301 CGTTGGTTCC TCGACAAGCT GGTGGACGAG ATCGGGGTGG GCTACGTGTA CGACCGCGGC   361 AGCGTCTCCG ACTACTATCT GAGCGAGATC AAGCCTCTGC ACAACTTCCT GACCCAGCTC   421 CAGCCCTTCC TGAAGCTCAA GCAGAAGCAG GCCAACCTCG TGCTGAAGAT CATCGAGCAG   481 CTGCCCTCCG CCAAGGAATC CCCGGACAAG TTCCTGGAGG TGTGCACGTG GGTGGACCAG   541 ATCGCGGCCC TCAACGACAG CAAGACCCGC AAGAGGACCT CGGAGACGGT GCGAGCGGTC   601 CTGGACTCCC TCCCAGGATC CGTGGGAGGT CTATCGCCAT CTCAGGCATC CAGCGCCGCA   661 TCCTCGGCTT CCTCAAGCCC GGGTTCAGGG ATCTCCGAAG CACTCAGAGC TGGAGCAGGT   721 TCCGGCACTG GATACAACAA GGAATTCCTG CTCTACCTGG CGGGCTTCGT GGACGGGGAC   781 GGCTCCATCT GGGCCCGGA TCAAGCCGGG GCAGTCCTAC AAGTTCAAGC ATACCCTGGAG   841 CTCGTGTTCC AGGTCACCCA GAAGACACAG CGCCGTTGGA TCCTCGACAA GCTGGTGGAC   901 GAGATCGGGG TGGGCTACGT GACCGACGCC GGCAGCGCCT CCGTCTACCG CCTGAGCGAG   961 ATCAAGCCTC TGCACAACTT CCTGACCCAG CTCCAGCCCT TCCTGAAGCT CAAGCAGAAG  1021 CAGGCCAACC TCGTGCTGAA GATCATCGAG CAGCTGCCCT CCGCCAAGGA ATCCCCGGAC  1081 AAGTTCCTGG AGGTGTGCAC CTGGGTGGAC CAGATCGCCG CTCTGAACGA CTCCAAGACC  1141 CGCAAGACCA CTTCCGAGAC CGTCCGCGCC GTTCTAGACA GTCTCTCCGA GAAGAAGAAG  1201 TCGTCCCCCT AGACAGTCTC TCCGAGAAGA AGAAGTCGTC CCCCTAGCGG CCGCTTCGAG  1261 CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA  1321 AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA  1381 ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG GGGGAGATGT  1441 GGGAGGTTTT TTAAAGCAAG TAAAACCTCT ACAAATGTGG TAAAATCGAT AAGATCTTGA  1501 TCCGGGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG  1561 CCTGAATGGC GAATGGACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT  1621 ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC  1681 CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT  1741 TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT  1801 GGTTCACGTA GTGGGCCATC G (Forward PCR primer for evaluating CGS-5/6 target site) SEQ ID NO: 23     1 tgacagctct ggccttaagt gcctacgaaa ctag (Reverse PCR primer for evaluating CGS-5/6 target site) SEQ ID NO: 24     1 gtctttcctc tttgctgtag ccttggtaga actactgcc (CHO-23/24 Insertion target sequence donor plasmid) SEQ ID NO: 25     1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA    61 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG   121 TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC   181 ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC   241 ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT   301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA AGGCCAGGGT   361 TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CCATACCCAG GGGAGCTGTA   421 CTGGGCTGCA GCCCTGCGCC ATTCAGCCAT GCACCAGGCT ACTCCCTCCT CTTCCAGCTT   481 TCTCCTTCTG ATGGCCATAG GATTAGAAGA TAAGGGACTC TAGTGCAGGT CAACTGCTGA   541 CCAGTGTGAA AATGCACAGA CTACATGCTG GTAGATCAGC ACTTCAAACT ACTGTTCACC   601 ATCATCTCTG GAATAAGCAC TACATTTACA GGGTTCAAAC CTCAATGAAT ATAAACAAAC   661 AAAACACACC TCCCTTCCTT CACTGTCTCC CATTTCTTTG GTTCCCATCT CCACATAGAA   721 TTTATAATTA AAATTTCTAA GTATCTTTCC AGAAATACTT CACACATGTT ATAAGCAAAT   781 GTGCTTTTAA AGATACTATT TTAAATTATG AAAATGGTTA TATTAGTTGA GATAAAAGAA   841 TAGAATGGGA AGTTCCAGAA TTTAAGGCCT CATATGGATC CCAGCTGTGG AATGTGTGTC   901 AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC   961 TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC  1021 AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC  1081 CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT  1141 ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGGTATTCCA GAAGTAGTGA GGAGGCTTTT  1201 TTGGAGGCTA CCATGGAGAA GTTACTATTC CGAAGTTCCT ATTCTCTAGA AAGTATAGGA  1261 ACTTCAAGCT TGGCACTGGG TACCGCCAAG TTGACCAGTG CCGTTCCGGT GCTCACCGCG  1321 CGCGACGTCG CCGGAGCGGT CGAGTTCTGG ACCGACCGGC TCGGGTTCTC CCGGGACTTC  1381 GTGGAGGACG ACTTCGCCGG TGTGGTCCGG GACGACGTGA CCCTGTTCAT CAGCGCGGTC  1441 CAGGACCAGG TGGTGCCGGA CAACACCCTG GCCTGGGTGT GGGTGCGCGG CCTGGACGAG  1501 CTGTACGCCG AGTGGTCGGA GGTCGTGTCC ACGAACTTCC GGGACGCCTC CGGGCCGGCC  1561 ATGACCGAGA TCGGCGAGCA GCCGTGGGGG CGGGAGTTCG CCCTGCGCGA CCCGGCCGGC  1621 AACTGCGTGC ACTTCGTGGC CGAGGAGCAG GACTGACACC CGAGCGAAAA CGGTCTGCGC  1681 TGCGGGACGC GCGAATTGAA TTATGGCCCA CACCAGTGGC GCGGCGACTT CCAGTTCAAC  1741 ATCAGCCGCT ACAGTCAACA GCAACTGATG GAAACCAGCC ATCGCCATCT GCTGCACGCG  1801 GAAGAAGGCA CATGGCTGAA TATCGACGGT TTCCATATGG GGATTGGTGG CGACGACTCC  1861 TGGAGCCCGT CAGTATCGGC GGAATTCCAG CTGAGCGCCG GTCGCTACCA TTACCAGTTG  1921 GTCTGGTGTC AAAAATAATA ATAACCGGGC AGGGGGGATC TGCATGGATC TTTGTGAAGG  1981 AACCTTACTT CTGTGGTGTG ACATAATTGG ACAAACTACC TACAGAGATT TAAAGCTCTA  2041 AGGTAAATAT AAAATTTTTA AGTGTATAAT GTGTTAAACT ACTGATTCTA ATTGTTTGTG  2101 TATTTTAGAT TCCAACCTAT GGAACTGATG AATGGGAGCA GTGGTGGAAT GCCTTTAATG  2161 AGGAAAACCT GTTTTGCTCA GAAGAAATGC CATCTAGTGA TGATGAGGCT ACTGCTGACT  2221 CTCAACATTC TACTCCTCCA AAAAAGAAGA GAAAGGTAGA AGACCCCAAG GACTTTCCTT  2281 CAGAATTGCT AAGTTTTTTG AGTCATGCTG TGTTTAGTAA TAGAACTCTT GCTTGCTTTG  2341 CTATTTACAC CACAAAGGAA AAAGCTGCAC TGCTATACAA GAAAATTATG GAAAAATATT  2401 CTGTAACCTT TATAAGTAGG CATAACAGTT ATAATCATAA CATACTGTTT TTTCTTACTC  2461 CACACAGGCA TAGAGTGTCT GCTATTAATA ACTATGCTCA AAAATTGTGT ACCTTTAGCT  2521 TTTTAATTTG TAAAGGGGTT AATAAGGAAT ATTTGATGTA TAGTGCCTTG ACTAGAGATC  2581 ATAATCAGCC ATACCACATT TGTAGAGGTT TTACTTGCTT TAAAAAACCT CCCACACCTC  2641 CCCCTGAACC TGAAACATAA AATGAATGCA ATTGTTGTTG TTAACTTGTT TATTGCAGCT  2701 TATAATGGTT ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA  2761 CTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT CTTATCATGT CTGGATCCCC  2821 AGGAAGCTCC TCTGTGTCCT CATAAACCCT AACCTCCTCT ACTTGAGAGG ACATTCCAAT  2881 CATAGGCTGC CCATCCACCC TACTAGTATA TGAAAATATA AAGCGCTTTC TCTTTTAAGT  2941 CTAGGGTAGG TGTACTAGAT CAGCGCTCAG CTCCATACCA TGAAGCCATC CAGGAGTCAG  3001 ACCTCTCTGA CAGCCCTGCC ATTGTCACAG AGAAGTTTCT GTCACCAGTG CTCATGCTGT  3061 CAGAGGAGGG AAGGAGAAAA GATGTGAGAC CTCCCAAGTC AAAGTCATCT ATGGATAAAA  3121 CCTTAGTTGC ATGGCACACC AGTGTTAGGG AGTCGGGGAA ACACAGCCAT AGCCCAGCTT  3181 CCTCTCTGTT CTTGCTCTTA TTACCACCAG AAAGAGGTTG CTTAGACAAC CCAAACCAAG  3241 ACACAGGGCT CTGTGGGAGG GAATCAGTCC CAGGCTTCTG GCACATGCTA TGTCACCGGA  3301 AAGCCCCAGC CCTACTCCGA ATCCCCACAA GTACAGCAAA TATCAGATTA TAGCATTTAA  3361 AGGGGCACTC TTGCCAAAGA GAAGCACCAT TGGAATAGCC ATGCTTGAGA ACTAAGCTTG  3421 GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC  3481 AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC  3541 ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG  3601 CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT  3661 TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC  3721 TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA  3781 GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT  3841 AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC  3901 CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT  3961 GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG  4021 CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG  4081 GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT  4141 CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG  4201 ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC  4261 GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA  4321 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT  4381 GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT  4441 TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA  4501 TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC  4561 TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT  4621 ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA  4681 ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA  4741 CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA  4801 AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA  4861 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG  4921 GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA  4981 GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT  5041 GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT  5101 CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA  5161 TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT  5221 ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA  5281 AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC  5341 AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG  5401 CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC  5461 CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT  5521 GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA  5581 CCTGACGTCT AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG  5641 AGGCCCTTTC GTC (reverse PCR primer in the SV40 early promoter) SEQ ID NO: 26     1 AGATGCATGC TTTGCATACT TCTGCCTGC (donor plasmid for inserting GFP into FRT Insertion target sequence) SEQ ID NO: 27     1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG    61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG   121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC   181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT   241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA   301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC   361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC   421 ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT   481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT   541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA   601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG   661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC   721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG   781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA   841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC   901 GTTTAAACTT AAGCTTAGCC ACCaTGGTGA GCAAGGGCGA GGAGCTGTTC ACCGGGGTGG   961 TGCCCATCCT GGTCGAGCTG GACGGCGACG TAAACGGCCA CAAGTTCAGC GTGTCCGGCG  1021 AGGGCGAGGG CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC ACCACCGGCA  1081 AGCTGCCCGT GCCCTGGCCC ACCCTCGTGA CCACCCTGAC CTACGGAGTG CAGTGCTTCA  1141 GCCGCTACCC CGACCACATG AAGCAGCACG ACTTCTTCAA GTCCGCCATG CCCGAAGGCT  1201 ACGTCCAGGA GCGCACCATC TTCTTCAAGG AGGACGGCAA CTACAAGACC CGCGCCGAGG  1261 TGAAGTTCGA GGGCGACACC CTGGTGAACC GCATCGAGCT GAAGGGCATC GACTTCAAGG  1321 AGGACGGCAA CATCCTGGGG CACAAGCTGG AGTACAACTA CAACAGCCAC AACGTCTATA  1381 TCATGGCCGA CAAGGAGAAG AACGGCATCA AGGTGAACTT CAAGATCCGC CACAACATCG  1441 AGGACGGGAG CGTGCAGCTC GCCGACCACT ACCAGCAGAA CACCCCCATC GGCGACGGCC  1501 CCGTGCTGCT GCCCGACAAC CACTACCTGA GCACCCAGTC CGCCCTGAGC AAAGACCCCA  1561 ACGAGAAGCG CGATCACATG GTCCTGCTGG AGTTCGTGAC CGCCGCCGGG ATCACTCTCG  1621 GCATGGACGA GCTGTACAAG TAAGGATCCA CTAGTCCAGT GTGGTGGAAT TCTGCAGATA  1681 TCCAGCAGAG TGGCGGCCGC TCGAGTCTAG AGGGCCCGTT TAAACCCGCT GATCAGCCTC  1741 GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC  1801 CCTGGAAGGT GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG CATCGCATTG  1861 TCTGAGTAGG TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA  1921 TTGGGAAGAC AATAGGAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGCTT CTGAGGCGGA  1981 AAGAACCAGC TGGGGCTCTA GGGGGTATCC CCACGCGCCC TGTAGCGGCG CATTAAGCGC  2041 GGCGGGTGTG GTGGTTACGC GCAGCGTGAC CGCTACACTT GCCAGCGCCC TAGCGCCCGC  2101 TCCTTTCGCT TTCTTCCCTT CCTTTCTCGC CACGTTCGCC GGCTTTCCCC GTCAAGCTCT  2161 AAATCGGGGG CTCCCTTTAG GGTTCCGATT TAGTGCTTTA CGGCACCTCG ACCCCAAAAA  2221 ACTTGATTAG GGTGATGGTT CACGTACCTA GAAGTTCCTA TTCCGAAGTT CCTATTCTCT  2281 AGAAAGTATA GGAACTTCCT TGGCCAAAAA GCCTGAACTC ACCGCGACGT CTGTCGAGAA  2341 GTTTCTGATC GAAAAGTTCG ACAGCGTCTC CGACCTGATG CAGCTCTCGG AGGGCGAAGA  2401 ATCTCGTGCT TTCAGCTTCG ATGTAGGAGG GCGTGGATAT GTCCTGCGGG TAAATAGCTG  2461 CGCCGATGGT TTCTACAAAG ATCGTTATGT TTATCGGCAC TTTGCATCGG CCGCGCTCCC  2521 GATTCCGGAA GTGCTTGACA TTGGGGAATT CAGCGAGAGC CTGACCTATT GCATCTCCCG  2581 CCGTGCACAG GGTGTCACGT TGCAAGACCT GCCTGAAACC GAACTGCCCG CTGTTCTGCA  2641 GCCGGTCGCG GAGGCCATGG ATGCGATCGC TGCGGCCGAT CTTAGCCAGA CGAGCGGGTT  2701 CGGCCCATTC GGACCGCAAG GAATCGGTCA ATACACTACA TGGCGTGATT TCATATGCGC  2761 GATTGCTGAT CCCCATGTGT ATCACTGGCA AACTGTGATG GACGACACCG TCAGTGCGTC  2821 CGTCGCGCAG GCTCTCGATG AGCTGATGCT TTGGGCCGAG GACTGCCCCG AAGTCCGGCA  2881 CCTCGTGCAC GCGGATTTCG GCTCCAACAA TGTCCTGACG GACAATGGCC GCATAACAGC  2941 GGTCATTGAC TGGAGCGAGG CGATGTTCGG GGATTCCCAA TACGAGGTCG CCAACATCTT  3001 CTTCTGGAGG CCGTGGTTGG CTTGTATGGA GCAGCAGACG CGCTACTTCG AGCGGAGGCA  3061 TCCGGAGCTT GCAGGATCGC CGCGGCTCCG GGCGTATATG CTCCGCATTG GTCTTGACCA  3121 ACTCTATCAG AGCTTGGTTG ACGGCAATTT CGATGATGCA GCTTGGGCGC AGGGTCGATG  3181 CGACGCAATC GTCCGATCCG GAGCCGGGAC TGTCGGGCGT ACACAAATCG CCCGCAGAAG  3241 CGCGGCCGTC TGGACCGATG GCTGTGTAGA AGTACTCGCC GATAGTGGAA ACCGACGCCC  3301 CAGCACTCGT CCGAGGGCAA AGGAATAGCA CGTACTACGA GATTTCGATT CCACCGCCGC  3361 CTTCTATGAA AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA TGATCCTCCA  3421 GCGCGGGGAT CTCATGCTGG AGTTCTTCGC CCACCCCAAC TTGTTTATTG CAGCTTATAA  3481 TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA  3541 TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAT CATGTCTGTA TACCGTCGAC  3601 CTCTAGCTAG AGCTTGGCGT AATCATGGTC ATAGCTGTTT CCTGTGTGAA ATTGTTATCC  3661 GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAG TGTAAAGGCT GGGGTGCCTA  3721 ATGAGTGAGC TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA  3781 CCTGTCGTGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT  3841 TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG  3901 AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC  3961 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT  4021 GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGAGGAGCAT CACAAAAATC GACGCTCAAG  4081 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC  4141 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC  4201 TTCGGGAAGC GTGGCGCTTT CTCATAGGTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT  4261 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT  4321 ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC  4381 AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA  4441 GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA  4501 GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG  4561 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA  4621 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG  4681 GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG  4741 AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT  4801 AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT  4861 CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT  4921 GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG  4981 AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG  5041 TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT  5101 TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC  5161 CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT  5221 CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC  5281 AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA  5341 GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC  5401 GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA  5461 ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA  5521 ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG  5581 AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG  5641 AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT  5701 GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT  5761 TCCCCGAAAA GTGCCACCTG AGGTC (reverse PCR primer in the hygromycin-resistance gene) SEQ ID NO: 28     1 CAGAAACTTC TCGACAGACG TCGCGGTGAG (CHOX-45/46 amino acid sequence) SEQ ID NO: 29     1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GSICASIRPE QERKFKHRLV LRFEVTQKTQ    61 RRWFLDKLVD EIGVGYVYDS GSVSRYYLSQ IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE   121 QLPSAKESPD KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA 

1.-19. (canceled)
 20. A method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site proximal to a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5′ flanking region 5′ to the recognition sequence; and (iii) a 3′ flanking region 3′ to the recognition sequence; and (b) introducing a double-stranded break between the 5′ and 3′ flanking regions of the endogenous target site; (c) contacting the cell with a donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the endogenous target site; (ii) an exogenous sequence; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the endogenous target site; whereby the donor 5′ flanking region, the exogenous sequence and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the endogenous target site by homologous recombination to provide a modified cell.
 21. The method of claim 20, further comprising growing the modified cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene.
 22. The method of claim 20, wherein the exogenous sequence comprises a gene of interest.
 23. The method of claim 20, wherein the endogenous target site is downstream from the 3′ regulatory region of the selectable gene.
 24. The method of claim 23, wherein the endogenous target site is 0 to 100,000 base pairs downstream from the 3′ regulatory region of the selectable gene.
 25. The method of claim 20, wherein the endogenous target site is upstream from the 5′ regulatory region of the selectable gene.
 26. The method of claim 25, wherein the endogenous target site is 0 to 100,000 base pairs upstream from the 5′ regulatory region of the selectable gene.
 27. The method of claim 20, wherein the selectable gene is glutamine synthetase (GS) and the locus is methionine sulphoximine (MSX) amplifiable.
 28. The method of claim 20, wherein the selectable gene is dihydrofolate reductase (DHFR) and the locus is Methotrexate (MTX) amplifiable.
 29. The method of claim 20, wherein the selectable gene is selected from the group consisting of Dihydrofolate Reductase, Glutamine Synthetase, Hypoxanthine Phosphoribosyltransferase, Threonyl tRNA Synthetase, Na,K-ATPase, Asparagine Synthetase, Ornithine Decarboxylase, Inosine-5′-monophosphate dehydrogenase, Adenosine Deaminase, Thymidylate Synthetase, Aspartate Transcarbamylase, Metallothionein, Adenylate Deaminase (1,2), UMP-Synthetase and Ribonucleotide Reductase.
 30. The method of claim 29, wherein the selectable gene is amplifiable by selection with a selection agent selected from the group consisting of Methotrexate (MTX), Methionine sulphoximine (MSX), Aminopterin, hypoxanthine, thymidine, Borrelidin, Ouabain, Albizziin, Beta-aspartyl hydroxamate, alpha-difluoromethylornithine (DFMO), Mycophenolic Acid, Adenosine, Alanosine, 2′ deoxycoformycin, Fluorouracil, N-Phosphonacetyl-L-Aspartate (PALA), Cadmium, Adenine, Azaserine, Coformycin, 6-azauridine, pyrazofuran, hydroxyurea, motexafin gadolinium, fludarabine, cladribine, gemcitabine, tezacitabine and triapine.
 31. A method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site proximal to a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5′ flanking region 5′ to the recognition sequence; and (iii) a 3′ flanking region 3′ to the recognition sequence; and (b) introducing a double-stranded break between the 5′ and 3′ flanking regions of the endogenous target site; (c) contacting the cell with an engineered target site donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the endogenous target site; (ii) an exogenous sequence comprising an engineered target site; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the endogenous target site; whereby the donor 5′ flanking region, the exogenous sequence and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the endogenous target site by homologous recombination to provide a mammalian cell comprising the engineered target site; (d) introducing a double-stranded break between the 5′ and 3′ flanking regions of the engineered target site; (e) contacting the cell comprising the engineered target site with a sequence of interest donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the engineered target site; (ii) an exogenous sequence comprising a sequence of interest; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the engineered target site; whereby the donor 5′ flanking region, the exogenous sequence comprising the sequence of interest and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the engineered target site by homologous recombination to provide an engineered mammalian cell comprising the sequence of interest.
 32. The method of claim 31, further comprising growing the engineered mammalian cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene.
 33. (canceled)
 34. The method of claim 31, wherein the endogenous target site is downstream from the 3′ regulatory region of the selectable gene.
 35. (canceled)
 36. The method of claim 31, wherein the endogenous target site is upstream from the 5′ regulatory region of the selectable gene.
 37. (canceled)
 38. The method of claim 31, wherein the selectable gene is glutamine synthetase (GS) and the locus is methionine sulphoximine (MSX) amplifiable.
 39. The method of claim 31, wherein the selectable gene is dihydrofolate reductase (DHFR) and the locus is Methotrexate (MTX) amplifiable. 40.-54. (canceled)
 55. A recombinant meganuclease comprising a polypeptide having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9, wherein the meganuclease recognizes and cleaves a recognition site of SEQ ID NO:
 7. 56. The recombinant meganuclease of 55, having the sequence of the meganuclease of SEQ ID NO:
 9. 57-70. (canceled) 